Novel compound obtained from gamboge resin, and medical uses of the same

ABSTRACT

Disclosed herein are seventeen new compounds obtained from an acetone-extracted product of gamboge resin. The seventeen new compounds have activities in inhibiting the growth of tumor/cancer cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of Taiwanese Application No. 099119107,filed on Jun. 11, 2010.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to seventeen new compounds obtained from anacetone-extracted product of gamboge resin. The seventeen new compoundshave been demonstrated to have activities in inhibiting the growth oftumor/cancer cells. Therefore, this invention also relates to the use ofthe seventeen new compounds in the preparation of pharmaceuticalcompositions.

2. Description of the Related Art

Gamboge resin is the gum-resin secreted by the plant of Garcinia sp. ofthe family Guttiferae. It has been used as a source of vegetative dyesand pigments since the old days. It is also used in folk medicine insome areas such as India and Thailand.

Garcinia (TENGHUANG in pinyin), which is commonly known as gamboge, is akind of evergreen trees that grow in tropical regions. The main speciesgrown in India is “Garcinia morella Desv,” whereas the main speciesgrown in Thailand is “G. harburyi Hook f.” Before the flowering period,the bark of the tree is cut open in a spiral shape about 2 meters fromthe ground to collect the exuding resin. The resin is then subjected toheat-drying to result in a solidified gamboge resin.

According to traditional Chinese medicine (TCM), gamboge is effective incombating inflammations, clearing away toxins, stopping blood bleeding,and killing worms. Ever since 1934, there have been a number of reportson the components of the gamboge resin. At present, it is known thatmany compounds can be isolated from extracts of gamboge resin,including: morellin, morellic acid, gambogic acid, morellinol,isomorellin, isomorellic acid, isogambogic acid, isomorellinol,neogambogic acid, desoxymorellin, dihydroisomorellin, α-guttiferin,β-guttiferin, gambogenic acid, desoxygambogenin, gambogellic acid,epigambogic acid, epiisogambogic acid, isogambogenic acid,30-hydroxygambogic acid, etc.

Some studies have reported the cytotoxic activity of certain componentsof gamboge resin on human cervical cancer cells HeLa, humannasopharyngeal cancer cells KB, human leukemia cells K562, anddoxorubicin-resistant K562 cell lines, etc. (J. Asano et al. (1996),Phytochemistry, 41:815-820; L. J. Lin et al. (1993), Magnetic Resonancein Chemistry, 31:340-347; Q. B. Han et al. (2006), Planta Med.,72:281-284; Q. B. Han et al. (2006), Chem. Pharm. Bull., 54:265-267; Q.B. Han et al. (2006), Chemistry & Biodiversity, 3:101-105).

U.S. Pat. No. 6,462,041 B1 has disclosed gambogic acid and its analogsand derivatives as represented by the following Formulae I, II and III:

-   -   wherein a dash line denotes a single bond, a double bond, or an        epoxy group; and X, Y, and R₁ to R₃ have the definitions as        disclosed in said US patent.

Compounds having one of the aforesaid Formulae I-III are disclosed to beactivators of caspases and inducers of apoptosis. However. U.S. Pat. No.6,462,041 B1 has disclosed neither a compound of Formula I that has a32,33-epoxy group, nor a process for preparing the same.

In Acta Chimica Sinica, 2008, 66(22):2513-2517, Ming-Meia Jia et al.studied the chemical components of Garcinia hanburyi and isolatedfifteen compounds from the cold ethanol-extracted product of Garciniahanburyi via silica gel column chromatography (gradient elution solventsystem: petroleum ether-acetone, acetone, methanol) and preparativeHPLC. According to structure indentification, the fifteen compounds wereidentified as 2α-hydroxy-3β-acetoxy-Iup-20(29)-en-28-oic acid (1),10α-hydroxyepigambogic acid (2), gambogic acid (3), isogambogic acid(4), gambogin (5), gambogoic acid B (6), desoxymorellin (7), isomorellin(8), gambogenic acid (9), isogambogenin (10), gambogellic acid (11),desoxygambogenin (12), morellic acid (13), isomorellic acid (14), and30-hydroxygambogic acid (15).

U.S. Pat. No. 7,138,428 B2 (corresponding to TW I282280 and CN 100413868C) has disclosed an acetone-extracted product from gamboge resin, i.e.,TSB-14. In addition, nine compounds were further purified from saidacetone-extracted product TSB-14, including a new compound formoxanthoneA, and eight known compounds betulin, betulinic acid, morellic acid,isomorellic acid, gambogic acid, isogambogic acid, isomorellinol anddesoxymorellin.

The acetone-extracted product TSB-14 and the nine purified compoundshave been demonstrated to have effects in inhibiting the growth oftumor/cancer cells such as liver cancer cells (HepG2), lung cancer cells(A549), breast cancer cells (MCF-7), colon cancer cells (HT-29),leukemia cells (HL-60), and lymphoma cancer cells (U937).

US 2007/0093456 A1 has disclosed a derivative of gambogic acid, which isidentified as “methyl 37,38-dihydroxy-gambogate” and has the followingchemical structure:

It has been found from experiments that methyl 37,38-dihydroxy-gambogatecan act as an activator of caspases and an inducer of apoptosis.

In Journal of Natural Products (2009), 72:117-124, S. J. Tao et al.reported that twelve new xanthones (such as oxygambogic acid shownbelow, methyl 8,8a-dihydromorellate, 7-methoxygambogellic acid, etc.)and a pair of new natural products (i.e., 8,8a-dihydro-8-hydroxygambogicacid and its isomer) were isolated from the resin of G. hanbury.

The stereostructure of oxygambogic acid has yet to be confirmed sincethe configuration of the C₁₅/C₁₆ double bond could not be determined dueto the overlapping of the H-15 and H-16 signals in the ¹H-NMR spectraldata. S. J. Tao et al. further found from pharmacological experimentsthat in addition to methyl 8,8a-dihydromorellate, all of the othertested thirteen compounds were effective in inhibiting the growth ofHeLa cancer cells.

In addition to the activities in inhibiting the growth of tumor/cancercells, the extracts of gamboge resin have been demonstrated to haveother biological activities. For instance, in the report of A. Panthonget al. Journal of Ethnopharmacology (2007), 111:335-340, an ethylacetate extract obtained from the resin of G. hanburyi Hook f. and namedGH5763 was demonstrated to exhibit anti-inflammatory, analgesic andantipyretic activities.

Despite the aforesaid, there still exists a need for medicinal chemistsand pharmaceutical manufacturers in the pharmaceutical industry toexplore new compound(s) or new extract(s) that can be easily preparedand that exhibit desirable biological activities such as anti-canceractivity, analgesic activity, anti-inflammatory activity, etc.

Upon further investigation, the applicants have obtained seventeen newcompounds from the acetone-extracted product TSB-14 of gamboge resin, inwhich the seventeen new compounds have been demonstrated to haveanti-cancer activity.

SUMMARY OF THE INVENTION

Therefore, according to a first aspect, this invention provides acompound purified from gamboge resin and selected from the groupconsisting of:

(1) a compound of the formula:

(2) a compound of the formula:

(3) a compound of the formula:

(4) a compound of the formula:

(5) a compound of the formula:

(6) a compound of the formula:

(7) a compound of the formula:

(8) a compound of the formula:

(9) a compound of the formula:

(10) a compound of the formula:

(11) a compound of the formula:

(12) a compound of the formula:

(13) a compound of the formula:

(14) a compound of the formula:

(15) a compound of the formula:

(16) a compound of the formula:

and

(15) a compound of the formula:

According to a second aspect, this invention provides a pharmaceuticalcomposition comprising one or more of the seventeen compounds describedabove.

According to a third aspect, this invention provides an anti-cancercomposition comprising one or more of the seventeen compounds describedabove.

According to a fourth aspect, this invention provides a method ofinhibiting the growth of tumor/cancer cells, comprising contacting thecells with one or more of the seventeen compounds described above.

According to a fifth aspect, this invention provides a method oftreating a cancer in a subject, comprising administering to the subjectone or more of the seventeen compounds described above.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of this inventionwill become apparent with reference to the following detaileddescription and the preferred embodiments taken in conjunction with theaccompanying drawings, in which:

FIG. 1 shows the analytical RP-HPLC elution profile of anacetone-extracted product from gamboge resin, which is referred to as“TSB-14” hereinafter and which was prepared according to the methoddisclosed in Example 1 of U.S. Pat. No. 7,138,428 B2, in which peaks1-35 respectively correspond to the thirty-five major componentsdiscovered during the retention time from 0 to 80 minutes;

FIG. 2 shows the elution profile of the product TSB-14 obtained from thesemi-preparative RP-HPLC analysis conducted in section A, entitled“Semi-preparative RP-HPLC analysis of the product TSB-14,” of Example 2,infra, in which the observed peaks 1-35 correspond to those shown in ofFIG. 1, respectively;

FIG. 3 shows the semi-preparative RP-HPLC elution profile of fraction 1obtained in section B, entitled “Preparation of fractions 1-3” ofExample 2, infra, in which the observed twelve peaks correspond to peaks1-12 shown in FIG. 2, respectively;

FIG. 4 shows the semi-preparative RP-HPLC elution profile of fraction 2obtained in Example 2, infra, in which the observed twelve peakscorrespond to peaks 13-24 shown in FIG. 2, respectively; and

FIG. 5 shows the semi-preparative RP-HPLC elution profile of fraction 3obtained in Example 2, infra, in which the observed eleven peakscorrespond to peaks 25-35 shown in FIG. 2, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood that, if any prior art publication is referred toherein, such reference does not constitute an admission that thepublication forms a part of the common general knowledge in the art, inTaiwan or any other country.

For the purpose of this specification, it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. One skilled in the art will recognize manymethods and materials similar or equivalent to those described herein,which could be used in the practice of this invention. Indeed, thisinvention is in no way limited to the methods and materials described.For clarity, the following definitions are used herein.

As used herein, the term “active component” or “biologically activecompound” is understood to include any substance or material, orcombination of substances and materials, which is pharmacologicallyactive and, hence, has therapeutic value.

In Example 1 of U.S. Pat. No. 7,138,428 B2, the applicants disclosed anacetone-extracted product from gamboge resin, i.e., TSB-14, which wasobtained by pulverizing gamboge resin into powder, followed byextracting the pulverized powder with acetone. The acetone-extractedproduct TSB-14 was further identified to contain formoxanthone A,betulin, betulinic acid, morellic acid, isomorellic acid, gambogic acid,isogambogic acid, isomorellinol, and desoxymorellin. In order to exploreany other biologically active component(s) that might exist in theacetone-extracted product TSB-14, in this invention, the applicantsemployed analytical RP-HPLC (reversed phase high performance liquidchromatography) and semi-preparative RP-HPLC analyses to isolate andpurify any possible new biologically active compound(s) existing in theacetone-extracted product TSB-14.

Firstly, the acetone-extracted product TSB-14 was subjected to ananalytical RP-HPLC analysis using an analytical RP-C8 column (LunaC8(2)). As shown in FIG. 1, the obtained analytical RP-HPLC elutionprofile was observed to have 35 major peaks, which were designated aspeaks 1 to 35, respectively.

The acetone-extracted product TSB-14 was then subjected to asemi-preparative RP-HPLC using a semi-preparative RP-C12 column (Synergi4μ C12). The obtained semi-preparative RP-HPLC elution profile as shownin FIG. 2, which was found to have 35 major peaks comparativelycorresponding to those shown in FIG. 1, was divided into three regions,in which Region 1 included peaks 1-12 discovered during the retentiontime from 0 to 42 minutes; Region 2 included peaks 13 to 24 discoveredduring the retention time from 42 to 135 minutes; and Region 3 includespeaks 25 to 35 discovered during the retention time from 135 to 280minutes.

Based on the three divided regions shown in FIG. 2, theacetone-extracted product TSB-14 was fractionated by semi-preparativeRP-HPLC to provide three eluates that respectively corresponded to saidthree divided regions. The three eluates thus collected wererespectively subjected to the following treatment: partitioning acollected eluate in H₂O and ethyl acetate; after washing with H₂O so asto remove TFA, the organic layer was dried on anhydrous Na₂SO₄,filtered, and the organic solvent was removed using a vacuum rotatoryevaporator. Three corresponding fractions 1-3 thus obtained wereseparately subjected to the semi-preparative RP-HPLC analysis to provideelution profiles as shown in FIGS. 3, 4 and 5, which were observed tohave peaks 1-12, 13-24 and 25-35, respectively.

The obtained fraction 1 was subjected to the semi-preparative RP-HPLCanalysis and, according to its elution profile shown in FIG. 3, twelveelates that respectively corresponded to peaks 1-12 shown in FIG. 3 werecollected. The collected eluates 1-12 were then subjected to thefollowing treatment: partitioning a collected eluate in H₂O and ethylacetate; after washing with H₂O so as to remove TFA, the organic layerwas dried on anhydrous Na₂SO₄, filtered, and the organic solvent wasremoved using a vacuum rotatory evaporator. Twelve crude products wereobtained, each of which was further purified. After completion ofpurification, twelve purified products were obtained from the eluates1-12 of fraction 1 and were designated as Gh-3261, Gh-3271, Gh-3272,Gh-3311, Gh-3332, Gh-1036, Gh-3291, Gh-631, Gh-1052, Gh-3351, Gh-3353and Gh-3352, respectively (see Table 1 of Example 2, infra).

Fraction 2 was treated in a manner identical to fraction 1, andaccording to its elution profile shown in FIG. 4, eluates 13-24 thatrespectively corresponded to peaks 13-24 shown in FIG. 4 were collected,and twelve purified products were further obtained from the eluates13-24 of fraction 2 and were designated as Gh-47, Gh-4602, Gh-4601,Gh-1601-A, Gh-1050, Gh-1602, Gh-1631, Gh-2641-1, Gh-2501, Gh-2642,Gh-2507 and Gh-2505, respectively (see Table 2 of Example 2, infra).

Fraction 3 was likewise treated in a manner identical to fraction 1, andaccording to its elution profile shown in FIG. 5, eluates 25-35 thatrespectively corresponded to peaks 25-35 shown in FIG. 5 were collected,and eleven purified products were further obtained from the eluates25-35 of fraction 3 and were designated as Gh-2508, Gh-2603-1,Gh-2603-2, Gh-1641, Gh-1642, Gh-2605, Gh-2606, Gh-2607-B, Gh-2607-1A,Gh-2301 and Gh-4301, respectively (see Table 3 of Example 2, infra)

The thirty-five products purified from fractions 1-3 were subjected tophysical and chemical analyses, including melting point determination,nuclear magnetic resonance (NMR) spectroscopy (e.g., ¹H-NMR and ¹³C-NMRspectroscopy), mass spectrometry (e.g., electron impact massspectrometry (EIMS), high-resolution electron impact mass spectrometry(HREIMS), fast atom bombardment mass spectrometry (FABMS), andhigh-resolution fast atom bombardment mass spectrometry (HRFABMS),etc.), etc.

Product Gh-631 has been confirmed by chemical structure analysis to be aknown compound, i.e., “formoxanthone A” disclosed in U.S. Pat. No.7,138,428 B2, which has the following chemical structure:

Products Gh-4602 and Gh-47 have been confirmed by chemical structureanalysis to be two known morellic acid stereoisomers, i.e., morellicacid and isomorellic acid, which have the following chemical structures,respectively:

Product Gh-4601 has been confirmed by chemical structure analysis to bea known derivative of isomorellic acid, i.e., isomorellinol, which hasthe following chemical structure:

Products Gh-1601-A and Gh-1602 have been confirmed by chemical structureanalysis to be two known C-2 epimers of 30-hydroxygambogic acid, i.e.,30-hydroxygambogic acid and 30-hydroxyepigambogic acid, which have thefollowing chemical structures, respectively:

Product Gh-2641-1 has been confirmed by chemical structure analysis tobe neogambogic acid, which has the following chemical structure:

Product Gh-2501 has been confirmed by chemical structure analysis to beisomorellin, which has the following chemical structure:

Products Gh-2505 and Gh-2642 have been confirmed by chemical structureanalysis to be two known gambogenic stereoisomers, i.e., gambogenic acidand isogambogenic acid, which have the following chemical structures,respectively:

Products Gh-2603-2 and Gh-2603-1 have been confirmed by chemicalstructure analysis to be two known C-2 epimers of gambogellic acid,i.e., gambogellic acid and epigambogellic acid, which have the followingchemical structures, respectively:

Products Gh-1641 and Gh-1642 have been confirmed by chemical structureanalysis to be two known C-2 epimers of isogambogic acid, i.e.,isogambogic acid and epiisogambogic acid, which have the followingchemical structures, respectively:

Products Gh-2605 and Gh-2606 have been confirmed by chemical structureanalysis to be two known C-2 epimers of gambogic acid, i.e., gambogicacid and epigambogic acid, which have the following chemical structures,respectively:

Product Gh-2301 has been confirmed by chemical structure analysis to bea known compound, i.e., desoxymorellin, which has the following chemicalstructure:

Product Gh-4301 has been confirmed by chemical structure analysis to bea known compound, i.e., desoxygambogenin, which has the followingchemical structure:

It has been confirmed from the results of chemical structure analysisand a comparison with the spectroscopic data of known compounds thatproducts Gh-3352 and Gh-3351 are two novel compounds which are hithertonot reported. Products Gh-3352 and Gh-3351, which are C-2 epimers withthe following chemical structures, are herein named as “formoxanthone E”and “epiformoxanthone E,” respectively:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds thatproducts Gh-1052 and Gh-1036 are two novel compounds which are hithertonot reported. Products Gh-1052 and Gh-1036, which are C-2 epimers withthe following chemical structures, are herein named as “formoxanthone F”and “epiformoxanthone F,” respectively:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds thatproducts Gh-3353 and Gh-3311 are two novel compounds which are hithertonot reported. Products Gh-3353 and Gh-3311, which, are C-2 epimers withthe following chemical structures, are herein named as “formoxanthone G”and “epiformoxanthone G,” respectively:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds thatproducts Gh-3261 and Gh-3271 are two novel compounds which are hithertonot reported. Products Gh-3261 and Gh-3271, which are C-2 epimers withthe following chemical structures, are herein named as “formoxanthone J”and “epiformoxanthone J,” respectively:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds that productGh-3272 is a novel compound which is hitherto not reported. ProductGh-3272 is herein named as “formoxanthone H” and has the followingchemical structure:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds that productGh-3332 is a novel compound which is hitherto not reported. ProductGh-3332 is herein named as “isoformoxanthone I” and has the followingchemical structure:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds that productGh-3291 is a novel compound which is hitherto not reported. ProductGh-3291 is herein named as “formoxanthone D” and has the followingchemical structure:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds that productGh-1631 is a novel compound which is hitherto not reported. ProductGh-1631 is herein named as “formoxanthone C” and has the followingchemical structure:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds that productGh-1050 is a novel compound which is hitherto not reported. ProductGh-1050 is herein named as “3α-hydroxygambogellic acid” and has thefollowing chemical structure:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds thatproducts Gh-2508 and Gh-2507 are two novel compounds which are hithertonot reported. Products Gh-2508 and Gh-2507, which are C-2 epimers withthe following chemical structures, are herein named as “β-gambogellicacid” and “β-epigambogellic acid,” respectively:

It has been confirmed from the result of chemical structure analysis anda comparison with the spectroscopic data of known compounds thatproducts Gh-2607-B and Gh-2607-1A are two novel compounds which arehitherto not reported. Products Gh-2607-B and Gh-2607-1A are C-2 epimerswith the following chemical structures, are herein named as“formoxanthone B” and “epiformoxanthone B,” respectively:

After comparing the chemical structures of the seventeen new compoundsobtained from fractions 1-3, the applicants found that these seventeennew compounds share a common skeleton structure and may be representedby the following formula (I):

wherein:

R₁ and R₂ together form a moiety selected from the group consisting of:

R₃ is selected from the group consisting of: 3-methyl-2-butenyl,1-hydroxy-2,3-epoxy-3-methylbutyl, 2,3-epoxy-3-methylbutyl, and2,3-dihydroxy-3-methylbutyl; and

R₄ is selected from the group consisting of: 3-carboxyl-2Z-butenyl,3-carboxyl-2E-butenyl, and 3-carboxyl-1E-butenyl; but excludingcompounds of the following formulae:

According to the chemical structure shown in the above formula (I), theseventeen new compounds obtained in this invention may be chemicallysynthesized by methodologies well known to those skilled in the art.

In this invention, the seventeen new purified compounds obtained fromthe acetone-extracted product TSB-14 from gamboge resin were subjectedto in vitro anti-cancer tests and were found to exhibit inhibitoryactivities against the growth of tumor/cancer cells (such as humanbreast adenocarcinoma cells, human colon adenocarcinoma cells, humanpromyelocytic leukemia cells, human hepatocellular carcinoma cells,human lung carcinoma cells, and human histocytic lymphoma cells). It istherefore contemplated that the seventeen new purified compounds havepotential for use in cancer therapy.

Accordingly, this invention provides a compound purified from gambogeresin and selected from the group consisting of:

(1) a compound of the formula:

(2) a compound of the formula:

(3) a compound of the formula:

(4) a compound of the formula:

(5) a compound of the formula:

(6) a compound of the formula:

(13) a compound of the formula:

(8) a compound of the formula:

(9) a compound of the formula:

(10) a compound of the formula:

(15) a compound of the formula:

(12) a compound of the formula:

(13) a compound of the formula:

(14) a compound of the formula:

(15) a compound of the formula:

(16) a compound of the formula:

and

(17) a compound of the formula:

This invention further provides a pharmaceutical composition comprisingone or more of the seventeen compounds described above.

This invention also provides a method of inhibiting the growth oftumor/cancer cells, comprising contacting the cells with one or more ofthe seventeen compounds described above.

In addition, this invention further provides an anti-cancer compositioncomprising one or more of the seventeen compounds described above.

This invention also provides a method of treating a cancer in a subject,comprising administering to the subject one or more of the seventeencompounds described above.

The pharmaceutical composition according to this invention can beformulated into a dosage form suitable for parenteral, topical, or oraladministration using technology well known to those skilled in the art,which includes, but is not limited to, injections (e.g., sterile aqueoussolutions or dispersions), sterile powder, tablets, troches, pills,capsules, and the like.

The pharmaceutical composition according to this invention can beparenterally administered via one or more of the following routes:intravenous injection, intramuscular injection, and subcutaneousinjection.

In a preferred embodiment according to this invention, thepharmaceutical composition is formulated into a dosage form suitable fororal administration.

The pharmaceutical composition according to this invention canadditionally comprise a pharmaceutically acceptable carrier widelyemployed in the art of drug-manufacturing. For instance, thepharmaceutically acceptable carrier may include one or more of thefollowing agents: solvents, emulsifiers, suspending agents, decomposers,binding agents, excipients, stabilizing agents, chelating agents,diluents, gelling agents, preservatives, lubricants, absorption delayingagents, plasticizer, filling agents, disintegrants, surfactants,thickening agents, liposomes, and the like.

The dosage and the frequency of administration of the pharmaceuticalcomposition according to this invention may vary depending on thefollowing factors: the severity of the disease to be treated, the routeof administration, and the weight, age, physical condition and responseof the subject to be treated. For instance, the daily dosage of thepharmaceutical composition according to this invention may be 2.1 to 3.0mg per kilogram of the body weight, and may be administered in a singledose or in several doses.

The present invention will be described in more detail with reference tothe following examples, which are given for the purpose of illustrationonly and are not intended to limit the scope of the present invention.

EXAMPLES General Procedures

Melting point was determined using a micro melting-point apparatus(Yanaco, Japan).

EIMS spectra and HREIMS spectra were recorded on a MAT-95 XLhigh-resolution mass spectrometer.

FABMS spectra and HRFABMS spectra were recorded on a JEOL JMSSX SX 102Amass spectrometer or a Finnigan MAT 95 XL mass spectrometer.

¹H-NMR and ¹³C-NMR spectra, ¹H-¹H COSY (homonuclear correlationspectroscopy) spectra, HMQC (hetero nuclear multiple-quantum coherence)spectra, HMBC (heteronuclear multiple-bond coherence) spectra, and NOESY(nuclear Overhauser effect spectroscopy) spectra, were recorded on aBRUKER AM400 spectrometer, a VARIAN GEMINI-400 spectrometer, or a BRUKERADVANCE DMX-600 spectrometer, in which CDCl₃ or acetone-d₆ was used as asolvent for the NMR measurements, and the chemical shift (δ) isexpressed in ppm relative to a standard.

Analytical reversed-phase high performance liquid chromatography(RP-HPLC) was performed using the following instruments: a HitachiL-7400 UV detector, a Hitachi L-7100 pump, a Hitachi HPLC D-7000 system,a column oven (Super CO-150, Enshine, Taiwan), a guard column(SecurityGuard cartridge C8, size: 3.0 mm×4.0 mm, Phenomenex, USA), andan analytical column (Luna 3μ C8(2) 100 Å, size: 4.6 mm×150 mm,Phenomenex, USA). Operation conditions for analytical RP-HPLC were asfollows: a sample injection volume of 5 μL; mobile phase: 0.05%trifluoroacetic acid (TFA)_((aq.))/100% acetonitrile (35:65, v/v);column flow rate: 0.75 mL/min; column oven temperature: 35° C.; anddetection wavelength: 360 nm.

Semi-preparative RP-HPLC was performed using the following instruments:a Hitachi L-7400 UV detector, a Hitachi L-7150 pump, a Hitachi HPLCD-7000 system, a column oven (Super CO-150, Enshine, Taiwan), a guardcolumn (SecurityGuard Cartridge C12, size: 10 mm×10 mm, Phenomenex,USA), and an analytical column (Synergi 4μ C12 80 Å, size: 10 mm×150 mm,Phenomenex, USA). Operation conditions for semi-preparative RP-HPLC wereas follows: a sample injection volume of 250 μL; mobile phase, which wasselected depending on experiment; column flow rate: 4.5 mL/min; columnoven temperature: 27° C.; and detection wavelength: 360 nm.

Example 1 Analysis of an Acetone-Extracted Product from Gamboge Resin(TSB-14) by Analytical RP-HPLC

An acetone-extracted product from gamboge resin, i.e., TSB-14, which wasprepared according to Example 1 of U.S. Pat. No. 7,138,428 B2, wassubjected to an analytical RP-HPLC analysis, so as to determine thechemical constituent(s) contained therein.

Briefly, 1.0 mg of the product TSB-14 was dissolved in 1 mL of acetoneand then subjected to an analytical RP-HPLC analysis along the lines asdescribed in the previous section of “General Procedures,” and anelution profile as shown in FIG. 1 was obtained.

Referring to FIG. 1, 35 major peaks (numbered from 1 to 35) are presentin the elution profile of the product TSB-14 within the retention timeof 0 to 80 minutes. According to the results shown in FIG. 1, theapplicants postulated that it might be possible to isolate 35 compoundsfrom the product TSB-14. To verify this postulation, the product TSB-14was subjected to the following experiments.

Example 2 Preparation of Purified Compounds from the Product TSB-14 A.Semi-Preparative RP-HPLC Analysis of the Product TSB-14

3 g of the product TSB-14 was dissolved in 30 mL of acetone/acetonitrile(v/v=1:9) and then subjected to a semi-preparative RP-HPLC analysisalong the lines as described in the previous section of “GeneralProcedures,” in which a mobile phase containing 0.05% TFA_((aq.))/65%acetonitrile_((aq.))(35:65) was used. The elution profile thus obtainedis shown in FIG. 2.

After comparing the two elution profiles shown in FIGS. 1 and 2, themajor peaks 1-35 as identified in FIG. 1 were also identified in theelution profile of FIG. 2, which was subsequently divided into threeregions, i.e., Region 1, which includes peaks 1-12 eluted at a retentiontime of 0 to 42 minutes; Region 2, which includes peaks 13 to 24 elutedat a retention time of 42 to 135 minutes; and Region 3, which includespeaks 25 to 35 eluted at a retention time of 135 to 280 minutes.

B. Preparation of Fractions 1-3

3 g of the product TSB-14 was subjected to a semi-preparative RP-HPLCanalysis as described in the preceding section A, and three eluates thatrespectively correspond to Regions 1, 2 and 3 of the elution profileshown in FIG. 2 were collected and then respectively subjected to thefollowing treatment: partitioning a collected eluate in H₂O and ethylacetate; after washing with H₂O so as to remove TFA, the organic layerwas dried on anhydrous Na₂SO₄, filtered, and the organic solvent wasremoved using a vacuum rotatory evaporator.

After the aforesaid treatment of the three collected eluates, threecorresponding fractions 1-3 were obtained.

C. Semi-Preparative RP-HPLC Analysis of Fractions 1-3

100 mg of each of fractions 1-3 as obtained in the preceding section Bwas dissolved in 1 mL of acetone, followed by subjecting to asemi-preparative RP-HPLC analysis as described in the preceding sectionA. The elution profiles of fractions 1-3 thus obtained are shown inFIGS. 3-5.

After comparing FIGS. 3, 4 and 5 with FIG. 2, it can be clearly seenthat peaks 1-12, peaks 13-24 and peaks 25-35 are present in thesemi-preparative RP-HPLC elution profiles of fractions 1, 2 and 3,respectively.

D. Preparation of Purified Compounds from Fraction 1

100 mg of fraction 1 as obtained in the preceding section B wasdissolved in 1 mL of acetone, followed by subjecting to asemi-preparative RP-HPLC analysis as described in the preceding sectionA, in which eluates 1-12 that respectively correspond to the elutionpeaks 1-12 in the semi-preparative RP-HPLC elution profile of fraction 1as shown in FIG. 3, were collected.

In order to obtain eluates 1-12 in large amount, elution of fraction 1by the semi-preparative RP-HPLC analysis as described above was repeatedfor 50 to 100 times. Eluates 1-12 thus collected were individuallyplaced into a 4 L brown glass bottle, followed by concentrating in avacuum rotatory evaporator, to thereby give concentrates 1-12, each ofwhich was in turn treated as follows: partitioning a concentrate in H₂Oand ethyl acetate; after washing with H₂O so as to remove TFA, theorganic layer was dried on anhydrous Na₂SO₄, filtered, and the organicsolvent was removed using a vacuum rotatory evaporator.

After the aforesaid treatment of concentrates 1-12, twelve crudeproducts were obtained, each of which was further purified as follows:100 mg of a crude product was dissolved in 1.0 mL of acetone and thensubjected to an analytical RP-HPLC analysis along the lines as describedin the previous section of “General Procedures,” so that an analyticalRP-HPLC elution profile containing only a single elution peak wasobtained. If the obtained elution profile failed to show the presence ofa single peak, the remainder of said crude product was subjected tofurther purification by repeating the above-described treatments untilan analytical RP-HPLC elution profile showing a single peak wasobtained.

After completion of purification, twelve purified products were obtainedfrom the eluates 1-12 of fraction 1 and were designated as Gh-3261,Gh-3271, Gh-3272, Gh-3311, Gh-3332, Gh-1036, Gh-3291, Gh-631, Gh-1052,Gh-3351, Gh-3353 and Gh-3352, respectively. The obtained weights ofthese twelve purified products are summarized in Table 1.

TABLE 1 The weights of twelve purified products from fraction 1.Eluate/Peak Product Weight (mg) 1 Gh-3261 9 2 Gh-3271 8 3 Gh-3272 7 4Gh-3311 7 5 Gh-3332 6 6 Gh-1036 10 7 Gh-3291 7 8 Gh-631 12 9 Gh-1052 610 Gh-3351 9 11 Gh-3353 7 12 Gh-3352 5E. Preparation of Purified Compounds from Fraction 2

100 mg of fraction 2 as obtained in the preceding section B wasdissolved in 1 mL of acetone, followed by subjecting to asemi-preparative RP-HPLC analysis as described in the preceding sectionA, in which eluates 13-24 that respectively correspond to the elutionpeaks 13-24 in the semi-preparative RP-HPLC elution profile of fraction2 as shown in FIG. 4, were collected.

In order to obtain eluates 13-24 in large amount, elution of fraction 2by the semi-preparative RP-HPLC analysis as described above was repeatedfor 50 to 100 times. Eluates 13-24 thus collected were individuallyplaced into a 4 L brown glass bottle, followed by concentrating in avacuum rotatory evaporator, to thereby give concentrates 13-24, each ofwhich was in turn subjected to partitioning separation and purificationaccording to the procedures as described in the preceding section D.

After completion of purification, twelve purified products were obtainedfrom the eluates 13-24 of fraction 2 and were designated as Gh-47,Gh-4602, Gh-4601, Gh-1601-A, Gh-1050, Gh-1602, Gh-1631, Gh-2641-1,Gh-2501, Gh-2642, Gh-2507 and Gh-2505, respectively. The obtainedweights of these twelve purified products are summarized in Table 2.

TABLE 2 The weights of twelve purified products from fraction 2.Eluate/Peak Product Weight (mg) 13 Gh-47 14 14 Gh-4602 48 15 Gh-4601 2716 Gh-1601-A 20 17 Gh-1050 16 18 Gh-1602 16 19 Gh-1631 19 20 Gh-2641-117 21 Gh-2501 13 22 Gh-2642 19 23 Gh-2507 8 24 Gh-2505 56F. Preparation of Purified Compounds from Fraction 3

100 mg of fraction 3 as obtained in the preceding section B wasdissolved in 1 mL of acetone, followed by subjecting to asemi-preparative RP-HPLC analysis as described in the preceding sectionA, in which eluates 25-35 that respectively correspond to the elutionpeaks 25-35 in the semi-preparative RP-HPLC elution profile of fraction3 as shown in FIG. 5, were collected.

In order to obtain eluates 1-25 in large amount, elution of fraction 3by the semi-preparative RP-HPLC analysis as described above was repeatedfor 50 to 100 times. Eluates 25-35 thus collected were individuallyplaced into a 4 L brown glass bottle, followed by concentrating in avacuum rotatory evaporator, to thereby give concentrates 25-35, each ofwhich was in turn subjected to partitioning separation and purificationaccording to the procedures as described in the preceding section D.

After completion of purification, eleven purified products were obtainedfrom the eluates 25-35 of fraction 3 and were designated as Gh-2508,Gh-2603-1, Gh-2603-2, Gh-1641, Gh-1642, Gh-2605, Gh-2606, Gh-2607-B,Gh-2607-1A, Gh-2301 and Gh-4301, respectively. The obtained weights ofthese eleven purified products are summarized in Table 3.

TABLE 3 The weights of eleven purified products from fraction 3. PeakProduct Weight (mg) 25 Gh-2508 18 26 Gh-2603-1 16 27 Gh-2603-2 18 28Gh-1641 20 29 Gh-1642 25 30 Gh-2605 162 31 Gh-2606 124 32 Gh-2607-B 1033 Gh-2607-1A 12 34 Gh-2301 14 35 Gh-4301 22

Example 3 Identification and Characterization of Compounds Isolated andPurified from Fractions 1-3

The physical and chemical properties of the thirty five productspurified as obtained from fractions 1-3 in the above Example 2 wereanalyzed according to the methodologies set forth in the precedingsection of “General Procedures,” including melting point (mp)measurement, ¹H-NMR, ¹³C-NMR, ¹H-¹H COSY, HMQC, HMBC, NOESY, EIMS,HREIMS, FABMS, and HRFABMS. The experimental data thus obtained aresummarized below.

1. Product Gh-47:

Product Gh-47, which was purified from eluate 13 of fraction 2, wasdetermined to have the following properties:

Yellow flake crystals; mp 204˜209° C.

EIMS m/z (relative intensity): 560 [M]⁺ (100), 545 (47), 532 (22), 517(36), 405 (44), 389 (11), 363 (24), 349 (17), 307 (12), 287 (22), 285(16), 245 (15), 215 (12), 189 (5).

¹H-NMR (400 MHz, CDCl₃): δ 7.56 (1H, d, J=6.9 Hz), 6.63 (1H, d, J=9.6Hz), 5.51 (1H, d, J=9.6 Hz), 5.12 (1H, dd, J=13.6, 6.8 Hz), 3.52 (1H,dd, J=6.2, 4.7 Hz), 3.26 (2H, d, J=7.4 Hz), 2.66 (1H, m), 2.55 (2H, m),2.35 (1H, dd, J=13.4, 4.6 Hz), 1.75 (3H, s), 1.72 (3H, s), 1.65 (3H, s),1.44 (3H, s), 1.43 (3H, s), 1.35 (3H, s), 1.30 (3H, s).

¹³C-NMR (100 MHz, CDCl₃): δ 200.93, 178.91, 171.86, 161.10, 157.64,157.28, 136.97, 135.39, 133.35, 131.70, 128.63, 126.18, 122.12, 115.44,108.27, 103.18, 100.49, 90.71, 83.71, 83.64, 78.65, 49.06, 46.88, 29.95,28.93, 28.33, 25.71, 25.31, 21.62, 18.09, 11.34.

According to the obtained spectral data, product Gh-47 was identified tobe a known compound having the following chemical structure, i.e.,isomorellic acid:

2. Product Gh-631:

Product Gh-631, which was purified from eluate 8 of fraction 1, wasdetermined to have the following properties:

Yellow needle crystals; mp 115˜118° C.

EIMS m/z (relative intensity): 394 [M]⁺ (66), 393 (23), 379 (100), 339(30), 323 (16), 311 (13), 295 (5), 278 (10), 203 (4), 162 (7).

¹H-NMR (600 MHz, acetone-d₆): δ 13.09 (1H, s, OH-1), 7.40 (1H, s, H-8),6.55 (1H, d, J=10.0 Hz, H-11), 6.32 (1H, s, H-2), 5.87 (1H, d, J=10.0Hz, H-12), 5.36 (H, m, H-17), 3.55 (2H, d, J=7.3 Hz, H-16), 1.84 (3H, s,H-20), 1.63 (3H, s, H-19), 1.48 (6H, s, H-14, H-15).

¹³C-NMR (150 MHz, acetone-d₆): δ 181.24 (C═O), 163.41 (C-3), 162.26(C-1), 155.56 (C-4a), 146.94 (C-6), 146.43 (C-10a), 134.42 (C-18),132.30 (C-12), 131.77 (C-5), 123.33 (C-17), 122.08 (C-11), 119.03 (C-7),115.20 (C-8a), 113.15 (C-8), 107.54 (C-4), 103.26 (C-9a), 98.40 (C-2),78.84 (C-13), 28.26 (C-14, 15), 25.93 (C-19), 22.16 (C-16), 18.00(C-20).

According to the obtained spectral data, product Gh-631 was identifiedto be a known compound having the following chemical structure, i.e.,formoxanthone A:

3. Product Gh-4601:

Product Gh-4601, which was purified from eluate 15 of fraction 2, wasdetermined to have the following properties:

Orange powder; mp 137˜139° C.

EIMS m/z (relative intensity): 546 [M]⁺ (100), 531 (18), 518 (44), 503(40), 485 (9), 433 (7), 405 (33), 391 (10), 363 (19), 349 (13), 307(10), 287 (25), 245 (8), 231 (18), 214 (12), 189 (5), 105 (6).

¹H-NMR (400 MHz, CDCl₃): δ 12.68 (1H, s), 7.41 (1H, d, J=7.2 Hz), 6.59(1H, d, J=10.0 Hz), 5.49 (1H, d, J=10.0 Hz), 5.19 (1H, t, J=7.0 Hz),4.73 (1H, t, J=8.0 Hz), 3.61 (2H, q, J=10.2 Hz), 3.49 (1H, d, J=4.7 Hz),3.47 (1H, d, J=4.7 Hz), 3.32 (1H, dd, J=14.5, 6.8 Hz), 3.24 (1H, dd,J=14.3, 7.7 Hz), 2.60 (1H, d, J=7.7 Hz), 2.48 (1H, d, J=9.4 Hz), 2.31(1H, dd, J=13.5, 4.7 Hz), 1.74, 1.68, 1.64, 1.25, 1.01 (each 3H, s),1.41 (6H, s).

¹³C-NMR (100 MHz, CDCl₃): δ 203.35, 180.33, 161.06, 157.80, 157.44,138.01, 134.40, 133.63, 131.87, 126.31, 121.90, 118.20, 115.50, 108.44,103.00, 100.72, 90.48, 84.50, 83.41, 78.66, 67.92, 49.09, 47.00, 30.09,28.96, 28.28, 28.21, 25.71, 25.29, 21.57, 18.14, 12.49.

According to the obtained spectral data, product Gh-4601 was identifiedto be a known compound having the following chemical structure, i.e.,isomorellinol:

4. Product Gh-4602:

Product Gh-4602, which was purified from eluate 14 of fraction 2, wasdetermined to have the following properties:

Orange powder; mp 106˜109° C.

EIMS m/z (relative intensity): 560 [M]⁺ (100), 545 (56), 532 (63), 517(48), 487 (12), 433 (9), 405 (81), 391 (22), 363 (38), 349 (24), 307(18), 287 (64), 245 (40), 231 (21), 215 (20), 189 (10).

¹H-NMR (400 MHz, CDCl₃): δ 12.71 (1H, s), 7.51 (1H, d, J=6.8 Hz), 6.49(1H, d, J=10.0 Hz), 6.05 (1H, t, J=7.0 Hz), 5.39 (1H, d, J=10.0 Hz),4.99 (1H, d, J=6.0 Hz), 3.45 (1H, dd, J=6.4, 4.7 Hz), 3.27 (1H, m), 3.08(1H, m), 2.97 (2H, sept, J=8.0 Hz), 2.49 (1H, d, J=9.3 Hz), 2.28 (1H,dd, J=13.4, 4.5 Hz), 1.70, 1.69, 1.67, 1.60, 1.36, 1.34, 1.26 (each 3H,s).

¹³C-NMR (100 MHz, CDCl₃): δ 203.47, 179.07, 171.74, 161.22, 157.65,157.34, 138.46, 135.39, 133.42, 131.46, 127.64, 126.00, 122.22, 115.44,108.04, 103.16, 100.55, 90.93, 83.82, 78.55, 49.01, 46.80, 29.87, 29.26,28.82, 28.40, 28.20, 25.68, 25.14, 21.57, 20.63, 18.06.

According to the obtained spectral data, product Gh-4602 was identifiedto be a known compound having the following chemical structure, i.e.,morellic acid:

5. Product Gh-2301:

Product Gh-2301, which was purified from eluate 34 of fraction 3, wasdetermined to have the following properties:

Orange needle crystals, mp 109˜110° C.

EIMS m/z (relative intensity): 530 [M]⁺ (100), 515 (22), 502 (92), 488(30), 487 (83), 459 (11), 433 (20), 405 (49), 391 (15), 363 (24), 349(16), 307 (13), 287 (27), 231 (13), 215 (37), 189 (6).

¹H-NMR (400 MHz, CDCl₃): δ 12.89 (1H, s), 7.45 (1H, d, J=7.2 Hz), 7.27(1H, s), 6.65 (1H, d, J=10.4 Hz), 5.53 (1H, d, J=9.6 Hz), 5.30 (1H, brd, J=6.0 Hz), 4.43 (1H, br s), 3.49 (1H, m), 3.32 (2H, m), 2.50 (2H, m),2.34 (1H, m), 1.78, 1.71, 1.68, 1.59, 1.33, 1.03 (each 3H, s), 1.45 (6H,s).

According to the obtained spectral data, product Gh-2301 was identifiedto be a known compound having the following chemical structure, i.e.,desoxymorellin:

6. Product Gh-4301:

Product Gh-4301, which was purified from eluate 35 of fraction 3, wasdetermined to have the following properties:

Yellow gummy solid; mp 85˜88° C.

EIMS m/z (relative intensity): 600 [M]⁺ (100), 572 (77), 557 (21), 531(10), 503 (60), 475 (26), 449 (33), 393 (9), 357 (12), 351 (26), 323(9), 309 (16), 295 (45), 281 (12), 253 (53), 231 (18), 215 (40), 189(10), 177 (18), 173 (12), 69 (65).

¹H-NMR (600 MHz, CDCl₃): δ 12.92 (1H, s), 7.41 (1H, d, J=7.0 Hz), 6.45(1H, s), 5.19 (2H, m), 5.03 (1H, m), 4.39 (1H, m), 3.45 (1H, dd, J=6.9,4.5 Hz), 3.36 (4H, m), 2.52 (2H, m), 2.43 (1H, d, J=9.4 Hz), 2.30 (1H,dd, J=13.5, 4.7 Hz), 2.05 (4H, m), 1.78 (3H, s), 1.74 (3H, s), 1.69 (3H,s), 1.654 (3H, s), 1.645 (3H, s), 1.56 (3H, s), 1.33 (3H, s), 1.30 (1H,m), 1.25 (3H, s), 0.98 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 203.55, 179.65, 162.98, 160.12, 156.29,139.04, 134.91, 133.88, 133.80, 133.75, 131.88, 123.74, 121.86, 121.33,117.76, 107.13, 106.39, 100.70, 90.12, 84.50, 83.11, 49.06, 46.90,39.66, 30.03, 29.02, 28.78, 26.32, 25.70, 25.63, 25.49, 25.40, 22.05,21.11, 18.01, 17.63, 16.68, 16.19.

According the obtained spectral data, product Gh-4301 was identified tobe a known compound having the following chemical structure, i.e.,desoxygambogenin:

7. Product Gh-2605:

Product Gh-2605, which was purified from eluate 30 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 205˜208° C.

EIMS m/z (relative intensity): 628 [M]⁺ (22), 600 (15), 545 (100), 517(23), 473 (17), 431 (7), 389 (9), 355 (10), 295 (5), 271 (7), 245 (14),215 (23), 189 (11), 69 (9).

¹H-NMR (600 MHz, CDCl₃): δ 12.73 (1H, s), 7.52 (1H, d, J=6.9 Hz), 6.57(1H, d, J=10.2 Hz), 6.08 (1H, dt, J=7.5, 1.3 Hz), 5.35 (1H, d, J=10.2Hz), 5.01 (2H, m), 3.46 (1H, dd, J=6.8, 4.6 Hz), 3.27 (1H, dd, J=14.7,8.1 Hz), 3.12 (1H, br dd, J=14.7, 5.3 Hz), 2.93 (2H, t, J=7.3 Hz), 2.49(1H, d, J=9.3 Hz), 2.29 (1H, dd, J=13.4, 4.7 Hz), 1.98 (2H, m), 1.72(3H, d, J=1.3 Hz), 1.71 (1H, m), 1.70 (3H, s), 1.67 (3H, s), 1.62 (3H,s), 1.60 (3H, s), 1.56 (1H, m), 1.52 (3H, s), 1.35 (3H, s), 1.34 (1H,m), 1.27 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 203.28, 178.85, 170.88, 161.49, 157.55,157.35, 137.90, 135.31, 133.34, 131.77, 131.49, 127.70, 124.49, 123.83,122.25, 115.87, 107.60, 102.73, 100.44, 90.91, 83.92, 83.78, 81.28,49.00, 46.81, 41.97, 29.85, 29.26, 28.84, 27.69, 25.64, 25.62, 25.16,22.73, 21.60, 20.73, 18.06, 17.60.

According the obtained spectral data, product Gh-2605 was identified tobe a known compound having the following chemical structure, i.e.,gambogic acid:

8. Product Gh-2606:

Product Gh-2605, which was purified from eluate 31 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 88˜92° C.

EIMS m/z (relative intensity): 628 [M]⁺ (24), 600 (11), 545 (100), 517(21), 473 (12), 431 (4), 389 (8), 347 (6), 245 (7), 215 (14), 189 (5),69 (4).

¹H-NMR (600 MHz, CDCl₃): δ 12.74 (1H, s), 7.53 (1H, d, J=6.9 Hz), 6.56(1H, d, J=10.0 Hz), 6.10 (1H, dt, J=7.4, 1.2 Hz), 5.38 (1H, d, J=10.0Hz), 5.07 (1H, br t, J=7.1 Hz), 5.01 (1H, br t, J=6.3 Hz), 3.45 (1H, dd,J=6.5, 4.9 Hz), 3.29 (1H, dd, J=14.6, 8.3 Hz), 3.13 (1H, br dd, J=14.6,4.3 Hz), 2.94 (1H, dd, J=16.1, 7.7 Hz), 2.87 (1H, dd, J=16.3, 6.3 Hz),2.49 (1H, d, J=9.3 Hz), 2.28 (1H, dd, J=13.4, 4.7 Hz), 2.05 (2H, m),1.75 (1H, m), 1.72 (3H, s), 1.71 (3H, s), 1.68 (3H, s), 1.64 (3H, s),1.62 (1H, m), 1.61 (3H, s), 1.55 (3H, s), 1.35 (1H, m), 1.31 (3H, s),1.26 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 203.25, 178.92, 170.68, 161.33, 157.59,157.33, 137.73, 135.51, 133.21, 131.99, 131.39, 127.76, 124.76, 123.82,122.24, 115.88, 107.79, 102.88, 100.51, 90.98, 83.86, 83.67, 81.10,49.00, 46.82, 41.69, 29.91, 29.26, 28.78, 26.91, 25.67, 25.60, 25.20,22.73, 21.61, 20.71, 18.12, 17.58.

According to the obtained spectral data, product Gh-2606 was identifiedto be a known compound having the following chemical structure, i.e.,epigambogic acid:

9. Product Gh-1641:

Product Gh-1641, which was purified from eluate 28 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 53˜56° C.

EIMS m/z (relative intensity): 628 [M]⁺ (32), 600 (6), 545 (100), 517(12), 473 (6), 431 (2), 389 (4), 355 (5), 245 (8), 215 (11), 189 (3), 69(5).

¹H-NMR (600 MHz, CDCl₃): δ 12.75 (1H, s), 7.53 (1H, d, J=6.9 Hz), 6.65(1H, d, J=10.1 Hz), 6.61 (1H, t, J=7.5 Hz), 5.41 (1H, d, J=10.1 Hz),5.09 (1H, t, J=6.9 Hz), 5.04 (1H, t, J=7.8 Hz), 3.49 (1H, dd, J=6.7, 4.6Hz), 3.24 (2H, m), 2.63 (1H, dd, J=15.6, 8.2 Hz), 2.53 (1H, m), 2.51(1H, d, J=9.3 Hz), 2.32 (1H, dd, J=13.5, 4.7 Hz), 2.02 (2H, dd, J=15.8,7.7 Hz), 1.76 (1H, m), 1.70 (3H, s), 1.69 (3H, s), 1.63 (3H, s), 1.62(3H, s), 1.59 (1H, m), 1.53 (3H, s), 1.38 (3H, s), 1.37 (1H, m), 1.34(3H, s), 1.28 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 202.97, 178.81, 171.55, 161.41, 157.61,157.35, 136.98, 135.31, 133.36, 131.81, 131.75, 128.56, 124.77, 123.82,122.18, 115.93, 107.92, 102.82, 100.40, 90.71, 83.73, 83.65, 81.33,49.05, 46.87, 41.92, 29.93, 29.05, 28.95, 27.49, 25.67, 25.61, 25.33,22.75, 21.61, 18.08, 17.59, 11.39.

According to the obtained spectral data, product Gh-1641 was identifiedto be a known compound having the following chemical structure, i.e.,isogambogic acid:

10. Product Gh-1642:

Product Gh-1642, which was purified from eluate 29 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 55˜60° C.

EIMS m/z (relative intensity): 628 [M]⁺ (63), 600 (13), 545 (100), 517(17), 473 (17), 431 (8), 389 (7), 355 (7), 245 (8), 215 (10), 189 (3),69 (18).

¹H-NMR (600 MHz, CDCl₃): δ 12.74 (1H, s), 7.52 (1H, d, J=7.1 Hz), 6.66(1H, d, J=10.2 Hz), 6.50 (1H, t, J=7.9 Hz), 5.44 (1H, d, J=10.2 Hz),5.12 (1H, t, J=6.9 Hz), 5.06 (1H, t, J=7.8 Hz), 3.49 (1H, dd, J=6.8, 4.5Hz), 3.25 (2H, m), 2.61 (2H, m), 2.49 (1H, d, J=9.5 Hz), 2.32 (1H, dd,J=13.3, 4.7 Hz), 2.06 (2H, m), 1.78 (1H, m), 1.73 (3H, s), 1.70 (3H, s),1.64 (3H, s), 1.63 (3H, s), 1.62 (1H, m), 1.55 (3H, s), 1.36 (3H, s),1.34 (1H, m), 1.28 (3H, s), 1.23 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 202.92, 178.88, 170.95, 161.32, 157.63,157.36, 136.75, 135.31, 133.33, 131.97, 131.74, 128.81, 124.81, 123.76,122.14, 115.96, 107.92, 102.93, 100.49, 90.64, 83.67, 83.62, 81.29,49.07, 46.95, 41.88, 30.03, 29.06, 28.98, 27.32, 25.71, 25.64, 25.48,22.75, 21.65, 18.16, 17.62, 11.43.

According to the obtained spectral data, product Gh-1642 was identifiedto be a known compound having the following chemical structure, i.e.,epiisogambogic acid:

11. Product Gh-2603-2:

Product Gh-2603-2, which was purified from eluate 27 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 131˜135° C.

EIMS m/z (relative intensity): 628 [M]⁺ (92), 600 (91), 545 (54), 517(46), 474 (100), 473 (88), 459 (18), 431 (29), 417 (15), 391 (33), 355(37), 349 (25), 295 (18), 253 (21), 245 (25), 215 (30), 189 (18), 69(20).

¹H-NMR (600 MHz, CDCl₃): δ 12.62 (1H, s, OH-6), 7.47 (1H, d, J=6.9 Hz,H-10), 6.00 (1H, dd, J=7.6, 1.3 Hz, H-27), 5.04 (1H, t, J=6.9 Hz, H-32),4.53 (1H, s, H₁-40), 4.19 (1H, s, H₂-40), 3.43 (1H, dd, J=13.4, 4.6 Hz,H-11), 3.40 (1H, br s, H-4), 3.23 (1H, dd, J=14.5, 8.1 Hz, H₁-31), 3.10(1H, dd, J=14.5, 5.6 Hz, H₂-31), 2.94 (2H, d, J=7.5 Hz, H-26), 2.51 (1H,d, J=9.3 Hz, H-22), 2.28 (1H, dd, J=13.4, 4.7 Hz, H₁-21), 2.08 (1H, brd, J=12.6 Hz, H-37), 1.88 (1H, br d, J=13.6 Hz, H₁-20), 1.82 (3H, s,H-39), 1.77 (1H, dd, J=13.1, 2.8 Hz, H₁-3), 1.71 (1H, m, H₂-3), 1.70(3H, s, H-34), 1.69 (3H, s, H-25), 1.68 (3H, s, H-29), 1.62 (3H, S,H-35), 1.52 (1H, dt, J=13.4, 4.9 Hz, H₂-20), 1.36 (1H, m, H₁-36), 1.33(1H, m, H₂-21), 1.30 (3H, s, H-19), 1.29 (1H, m, H₂-36), 1.27 (3H, s,H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 203.68 (C-12), 178.47 (C-8), 171.33 (C-30),164.44 (C-18), 160.77 (C-6), 155.27 (C-16), 148.87 (C-38), 138.06(C-27), 134.30 (C-10), 133.93 (C-9), 131.07 (C-33), 127.77 (C-28),122.44 (C-32), 108.60 (C-40), 106.26 (C-17), 104.14 (C-5), 99.57 (C-7),90.42 (C-14), 84.20 (C-23), 83.93 (C-13), 77.05 (C-2), 48.91 (C-22),48.10 (C-37), 46.73 (C-11), 39.27 (C-20), 36.52 (C-3), 29.90 (C-25),29.18 (C-26), 28.85 (C-4), 28.85 (C-24), 28.37 (C-19), 25.74 (C-35),25.18 (C-21), 22.96 (C-39), 22.78 (C-36), 21.84 (C-31), 20.71 (C-29),18.14 (C-34).

The EIMS data of Product Gh-2603-2 show a molecular ion peak [M]⁺ at m/z628, which is identical to that observed in gambogic acid. In addition,the recorded ¹H-NMR and ¹³C-NMR data of Product Gh-2603-2 were found tobe identical to those of a known compound, i.e., gambogellic acid, asfurther confirmed by the ¹H-¹H COSY, HMQC (J=150 Hz) and HMBC (J=8 Hz)spectral analyses (data not shown).

The NOESY data of Product Gh-2603-2 also reveal that S 7.47 (H-10) wascorrelated with δ 3.43 (H-11); δ 3.43 (H-11) was correlated with δ 2.28(H₁-21); δ 1.33 (H₂-21) was correlated with δ 2.51 (H-22); δ 2.28(H₁-21) was correlated with δ 2.51 (H-22) and δ 1.27 (H-24); and δ 6.00(H-27) was correlated with δ 1.68 (H-29), evidencing that thestereostructure of Product Gh-2603-2 in this part is identical to thatof gambogic acid. Namely, the stereostructure of Product Gh-2603-2 inthis part has a 11S,13R,14S,22S configuration, which includes H-27 andcarboxyl group (C-30) in a trans relationship, and a double bondΔ^(27,28) in a Z configuration.

In the monoterpene moiety of Product Gh-2603-2, the signal of themethine proton attached to the isopropenyl group (i.e., δ 2.08 (1H, brd, J=12.6 Hz, H-37)) reveals that the methine proton attached to theisopropenyl group and a vicinal proton have a relatively high vicinalcoupling constant (j=12.6 Hz) and, hence, they are in axial-axialcoupling. As such, H-37 is in an axial orientation.

It was further found from the NOESY data of Product Gh-2603-2 that: δ3.40 (H-4) was correlated not only with δ 2.08 (H-37), but also with δ1.77 (H₁-3) and δ 1.71 (H₂-3); δ 1.77 (H₁-3) was correlated with δ 1.30(H-19); δ 2.08 (H-37) was correlated with δ 1.77 (H₁-3) and δ 1.52(H₂-20); and δ 3.40 (H-4) was correlated with δ 1.68 (H-29). Since δ3.40 (H-4) was correlated with δ 1.68 (H-29), C-2 was in a Rconfiguration. The monoterpene moiety of Product Gh-2603-2 wasdetermined to have a rigid chair conformation, in which an 1,3-diaxialinteraction exists amongst H-37, H-3, and H-20; the methyl proton (H-19)attached to C-2 and the isopropenyl group attached to C-37 were in anequatorial orientation, so that the stereostructure of Product Gh-2603-2in this part has a 2R,4R,37S configuration.

Based on the above information, Product Gh-2603-2 was identified to be aknown compound having the following chemical structure, i.e.,gambogellic acid:

12. Product Gh-2603-1:

Product Gh-2603-1, which was purified from eluate 26 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 115˜120° C.

EIMS m/z (relative intensity): 628 [M]⁺ (92), 600 (100), 545 (68), 517(49), 474 (98), 473 (80), 459 (17), 431 (25), 417 (13), 391 (28), 355(24), 349 (18), 295 (11), 253 (12), 245 (13), 215 (16), 189 (7), 69(15).

¹H-NMR (600 MHz, CDCl₃): δ 12.68 (1H, s, OH-6), 7.48 (1H, d, J=6.9 Hz,H-10), 5.99 (1H, dt, J=8.0, 1.3 Hz, H-27), 5.00 (1H, brt, J=6.7 Hz,H-32), 4.52 (1H, s, H₁-40), 4.26 (1H, s, H₂-40), 3.50 (1H, br s, H-4),3.44 (1H, dd, J=6.8, 4.5 Hz, H-11), 3.31 (1H, dd, J=14.6, 8.2 Hz,H₁-31), 3.20 (1H, br dd, J=14.5, 3.8 Hz, H₂-31), 3.01 (1H, dd, J=16.5,7.6 Hz, H₁-26), 2.84 (1H, ddd, J=16.6, 7.1, 1.4 Hz, H₂-26), 2.46 (1H, d,J=9.4 Hz, H-22), 2.29 (1H, dd, J=13.5, 4.6 Hz, H₁-21), 2.13 (1H, br d,J=12.4 Hz, H-37), 1.92 (1H, br dd, J=13.8, 2.0 Hz, H₁-20), 1.86 (1H, dd,J=6.6, 2.7 Hz, H₁-3), 1.84 (3H, s, H-39), 1.70 (1H, m, H₂-3), 1.695 (3H,s, H-34), 1.69 (6H, s, H-25, H-29), 1.61 (3H, s, H-35), 1.54 (1H, dt,J=13.5, 5.2 Hz, H₂-20), 1.36 (2H, m, H-36), 1.34 (1H, m, H₂-21), 1.33(3H, s, H-19), 1.27 (3H, s, H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 203.23 (C-12), 178.53 (C-8), 170.69 (C-30),164.50 (C-18), 160.92 (C-6), 155.39 (C-16), 147.30 (C-38), 137.10(C-27), 134.59 (C-10), 133.70 (C-9), 131.11 (C-33), 128.00 (C-28),122.59 (C-32), 109.28 (C-40), 106.34 (C-17), 104.24 (C-5), 99.48 (C-7),90.48 (C-14), 83.90 (C-23), 83.61 (C-13), 77.09 (C-2), 49.13 (C-22),48.32 (C-37), 46.74 (C-11), 39.33 (C-20), 37.13 (C-3), 29.89 (C-25),29.50 (C-26), 29.09 (C-4), 28.95 (C-24), 28.52 (C-19), 25.69 (C-35),25.41 (C-21), 22.93 (C-39), 22.69 (C-36), 21.90 (C-31), 20.56 (C-29),18.16 (C-34).

The EIMS data of product Gh-2603-1 show a molecular ion peak [M]⁺ at m/z628, which reveals that the fragmentation pattern of Product Gh-2603-1is identical to that of Product Gh-2603-2 (i.e., gambogellic acid).Besides, the ¹H-NMR and ¹³C-NMR data of Product Gh-2603-1 are generallysimilar to those of Product Gh-2603-2.

The ¹H-NMR data of Product Gh-2603-1 show that the side chain connectedto C-13 has a proton signal different from that observed in acorresponding site of Product Gh-2603-2. That is, in Product Gh-2603-2the methylene proton in the 2-methyl-2-butenoic acid has a simpledoublet signal of δ 2.94 (2H, d, J=7.5 Hz, H-26), whereas in ProductGh-2603-1 the methylene proton in the 2-methyl-2-butenoic acid hassignals of δ 3.01 (1H, dd, J=16.5, 7.6 Hz, H₁-26) and δ 2.84 (1H, ddd,6.6, 7.1, 1.4 Hz, H₂-26). In the structure of gambogellic acid (ProductGh-2603-2) wherein C-2 is in a R configuration, the methylene group(C-26) is capable of free rotation. If C-2 is in a S configuration, themonoterpene p-menthene ring and the isopropenyl group containing C-2form a steric hindrance to free rotation, thereby resulting in anonequivalence of the two protons in the methylene group, as well as ananisotropic effect to relevant protons. Besides, the methine protonattached to the isopropenyl group, which has a signal of δ 2.13 (1H, brd, J=12.4 Hz, H-37), has a high coupling constant (J=12.4 Hz),indicating that the methine proton attached to the isopropenyl group isin an axial orientation.

The structure of Product Gh-2603-1 was further confirmed by the ¹H-¹HCOSY, HMQC and HMBC analyses, in which the HMBC data of the p-menthenemonoterpene show that the exo-methylene protons (δ 4.52 (H₁-40) and δ4.26 (H₂-40)) are correlated with δ 22.93 (C-39) and δ 48.32 (C-37); theC-3 methylene proton (δ 1.86 (H₁-3)) is correlated with δ 104.24 (C-5),δ 29.09 (C-4), δ 48.32 (C-37) and δ 147.30 (C-38); and the C-20methylene proton (δ 1.92 (H₁-20)) is correlated with δ 48.32 (C-37) andδ 29.09 (C-4). Moreover, the methyl proton (δ 1.33 (H-19)) attached toC-2 are correlated with two adjacent methylene carbons (δ 37.13 (C-3)and δ 39.33 (C-20)).

The HMBC data of Product Gh-2603-1 also show that: H-3 is correlatedwith C-5 in the aromatic ring, and both the isopropenyl group attachedto C-37 and the methyl proton (H-19) attached to C-2 are in anequatorial orientation. Therefore, the monoterpene moiety has a rigidchair conformation.

It is further found from the NOESY data of Product Gh-2603-1 that: δ2.13 (H-37) is correlated with δ 1.36 (H-36) and δ 1.54 (H₂-20). Thisindicates that a 1,3-diaxial interaction exists amongst the axial H-37and the two axial protons (H-3 and H-20) in the methylene group adjacentto C-2. Therefore, in contrast to Product Gh-2603-2, the p-menthene inProduct Gh-2603-1 is in a 2S,4S,37R configuration. In addition, theNOESY data of Product Gh-2603-1 also shows that: δ 7.48 (H-10) iscorrelated with δ 3.44 (H-11); δ 3.44 (H-11) is correlated with δ 2.29(H₁-21); δ 1.34 (H₂-21) is correlated with δ 2.46 (H-22); both δ 2.29(H₁-21) and δ 2.46 (H-22) are correlated with δ 1.27 (H-24); and δ 5.99(H-27) is correlated with δ 1.69 (H-29). These data evidence that thestereostructure of Product Gh-2603-1 at the right-side of the r-pyronethereof is identical to that of gambogic acid or gambogellic acid, i.e.,having a 11S,13R,14S,22S configuration, which includes H-27 and carboxylgroup (C-30) in a trans relationship, and a double bond Δ^(27,28) in a Zconfiguration.

Based on the above information, Product Gh-2603-1 was identified to be aknown compound having the following chemical structure, i.e.,“epigambogellic acid” {IUPAC nomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,9S,10R,13S,16aS)-3a,4,5,7,10,11,12,13-octahydro-8-hydroxy-3,3,13-trimethyl-15-(3-methyl-2-butenyl)-10-(1-methylethenyl)-7,18-dioxo-1,5:9,13-dimethano-1H,3H,9H-furo[3.4-g]oxocino[3.2-b]xanthen-1-yl]-(2Z)-]}:

13. Product Gh-2607-B:

Product Gh-2607-B, which was purified from eluate 32 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 120˜125° C.

EIMS m/z (relative intensity): 628 [M]⁺ (33), 600 (17), 545 (100), 517(23), 499 (4), 474 (14), 431 (5), 389 (8), 355 (7), 347 (6), 245 (5),215 (9), 189 (3), 69 (4); HREIMS [M]⁺ m/z: 628.3034; calculated forC₃₈H₄₄O₈, 628.3036.

¹H-NMR (600 MHz, CDCl₃): δ 12.57 (1H, s, OH-6), 7.50 (1H, d, J=6.9 Hz,H-10), 5.86 (1H, dt, J=7.6, 1.2 Hz, H-27), 5.10 (1H, brt, J=7.0 Hz,H-32), 3.46 (1H, dd, J=6.8, 4.5 Hz, H-11), 3.28 (1H, dd, J=14.7, 8.5 Hz,H₁-31), 3.15 (1H, m, H₁-26), 3.12 (1H, m, H₂-31), 2.95 (1H, d, J=9.6 Hz,H-3), 2.91 (1H, ddd, J=15.9, 6.9, 1.4 Hz, H₂-26), 2.50 (1H, d, J=9.3 Hz,H-22), 2.42 (1H, dd, J=9.5, 7.4 Hz, H-4), 2.31 (1H, m, H-37), 2.29 (1H,m, H₁-21), 1.74 (1H, m, H₁-20), 1.71 (3H, s, H-34), 1.69 (3H, s, H-25),1.67 (3H, s, H-29), 1.60 (3H, s, H-35), 1.60 (1H, m, H₁-36), 1.53 (1H,m, Hs-20), 1.49 (1H, m, H₂-36), 1.36 (1H, m, H₂-21), 1.30 (3H, s, H-40),1.28 (3H, s, H-19), 1.27 (3H, s, H-24), 0.71 (3H, s, H-39).

¹³C-NMR (150 MHz, CDCl₃): δ 203.71 (C-12), 178.89 (C-8), 171.22 (C-30),161.53 (C-18), 161.17 (C-6), 155.04 (C-16), 137.79 (C-27), 134.63(C-10), 133.88 (C-9), 130.85 (C-33), 128.20 (C-28), 122.37 (C-32),108.88 (C-17), 105.32 (C-5), 100.38 (C-7), 90.33 (C-14), 85.18 (C-2),84.18 (C-23), 83.86 (C-13), 48.94 (C-22), 46.81 (C-11), 46.23 (C-37),38.81 (C-38), 38.58 (C-20), 36.93 (C-4), 35.01 (C-3), 33.45 (C-40),29.91 (C-25), 29.25 (C-26), 28.93 (C-24), 27.35 (C-19), 25.74 (C-35),25.61 (C-36), 25.32 (C-21), 21.83 (C-31), 20.78 (C-29), 18.19 (C-34),17.68 (C-39).

The EIMS data of Product Gh-2607-B show a molecular ion peak [M]⁺ at m/z628 and the HREIMS data of Product Gh-2607-B show a molecular ion peak[M]⁺ at m/z 628.3034, indicating that Product Gh-2607-B has a molecularformula identical to those of the above-described compounds gambogicacid and gambogellic acid as well as their epimers, i.e., C₃₈H₄₄O₈.

The ¹H-NMR and ¹³C-NMR data of Product Gh-2607-B show that ProductGh-2607-B does not have a disubstituted double bond in the pyran ringthereof, nor does it have an isopropenyl group having a terminal doublebond.

As further confirmed by the ¹H-¹H COSY, HMQC and HMBC analyses, ProductGh-2607-B is structurally similar to gambogellic acid except for at themonoterpene moiety. As compared to gambogellic acid, Product Gh-2607-Bwas found to have one less double bond. Product Gh-2607-B was thereforepresumed to have one more ring than gambogellic acid.

The ¹H-¹H COSY data of Product Gh-2607-B shows that: a methine protonhaving a doublet signal (δ 2.95 (1H, d, J=9.6 Hz, H-3)) is coupled toonly another methine proton (δ 2.42 (1H, dd, J−9.5, 7.4 Hz, H-4)), whichis further coupled to a further methine proton (δ 2.31 (1H, m, H-37));the methine proton (δ 2.31 (1H, m, H-37)) is coupled to two methyleneprotons of C-36 (δ 1.60 (1H, m, H₁-36) and δ 1.49 (1H, m, H₂-36)); and afurther methylene proton (δ 1.74 (1H, m, H₁-20)) is coupled to the twomethylene protons of C-36 (δ 1.60 (1H, m, H₁-36) and δ 1.49 (1H, m,H₂-36)), evidencing that the monoterpene moiety of Product Gh-2607-Bstill has two adjacent methylene groups (C-36 (δ 25.61) and C-20 (δ38.58)) and three adjacent methine groups (C-3 (δ 35.01), C-4 (δ 36.93),and C-37 (δ 46.23)]). The methine group (C-3) of Product Gh-2607-B is atransformation of the methylene group (C-3) of gambogellic acid, andsaid transformation is presumed to be caused by forming a bond betweenC-3 and C-38 to replace a terminal double bond between C-38 and C-40,thereby resulting in the formation of a pinane-type monoterpenestructure, which includes a cyclobutane, a gem-dimethyl group (C-39 (δ17.68) and C-40 (δ 33.45)), an oxygen-bearing quaternary carbon (C-2 (δ85.18)), and a tertiary methyl group (C-19 (δ 27.35)) attached to theoxygen-bearing quaternary carbon.

The HMBC data of Product Gh-2607-B show that: in addition to beingcorrelated with an aromatic carbon δ 105.32 (C-5), δ 161.17 (C-6) and δ161.53 (C-18), δ 2.95 (H-3) is correlated with δ 85.18 (C-2), δ 46.23(C-37), δ 38.81 (C-38), δ 38.58 (C-20), δ 36.93 (C-4), δ 33.45 (C-40),and δ 17.68 (C-39); δ 2.42 (H-4) is correlated with δ 105.32 (C-5), δ85.18 (C-2), δ 38.81 (C-38), δ 35.01 (C-3), δ 27.35 (C-19) and δ 25.61(C-36); and δ 2.31 (H-37) is correlated with δ 85.18 (C-2), δ 38.81(C-38), δ 35.01 (C-3), δ 36.93 (C-4), and δ 33.45 (C-40). It can beknown from the above HMBC data that: in the pinane-type monoterpenestructure of Product Gh-2607-B, C-2 is connected to C-18 of an aromaticring via an ether bond; C-4 is connected to C-5; and the methyl group(C-19) attached to C-2 is in an equatorial orientation.

It is further found from the NOESY data of Product Gh-2607-B that: δ2.95 (H-3) is correlated with δ 2.42 (H-4), δ 1.28 (H-19) and δ 1.30(H-40); δ 2.42 (H-4) is correlated with δ 2.31 (H-37) and δ 1.30 (H-40);δ 2.31 (H-37) is correlated with δ 1.30 (H-40); δ 0.71 (H-39) iscorrelated with δ 1.30 (H-40), δ 1.28 (H-19), δ 1.74 (H₁-20) and δ 1.53(H₂-20); and δ 1.67 (H-29) is correlated with δ 5.86 (H-27), but thereis no cross peak between δ 1.67 (H-29), δ 2.42 (H-4) and δ 2.31 (H-37),indicating that C-2 is in an R configuration; the methyl group (C-19) isin a α-equatorial orientation; and in the pinane structure, the threemethine protons H-3, H-4 and H-37 in the cyclobutane ring are incis-relationship such that the cyclobutane ring is in a 3S,4S,37Rconfiguration.

The NOESY data of Product Gh-2607-B also reveal that: δ 7.50 (H-10) iscorrelated with δ 3.46 (H-11); δ 3.46 (H-11) is correlated with δ 2.29(H₁-21); δ 1.36 (H₂-21) is correlated with δ 2.50 (H-22); and δ 2.50(H-22) is correlated with δ 1.69 (H-25), evidencing that thestereostructure of Product Gh-2607-B in this part has a 11S,13R,14S,22Sconfiguration, which includes H-27 and a carboxyl group (C-30) in atrans-relationship inasmuch as δ 1.67 (H-29) is correlated with δ 5.86(H-27), and a double bond Δ^(27,28) in a Z configuration.

Based on the above information, Product Gh-2607-B is identified to be anew compound having the following chemical structure:

Product Gh-2607-B is identified by the name “formoxanthone B” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,9S,10S,12R,15R,18aS)-3a,4,5,7,12,13,14,15-octahydro-8-hydroxy-3,3,11,11,15-pentamethyl-17-(3-methyl-2-butenyl)-7,19-dioxo-1,5-methano-1H,3H,9H-furo[3.4-g]-16-oxa-tricyclo[4.4.0.0^(9,12)]decano[3.2-b]xanthen-1-yl]-(2Z)-]}.

14. Product Gh-2607-1A:

Product Gh-2607-1A, which was purified from eluate 14 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 146˜151° C.

EIMS m/z (relative intensity): 628 [M]⁺ (27), 601 (13), 545 (100), 517(20), 473 (12), 389 (8), 355 (7), 347 (7), 245 (10), 215 (18), 189 (8),69 (11); HREIMS [M⁺] m/z: 628.3046; calculated for C₃₈H₄₄O₈, 628.3036.

¹H-NMR (600 MHz, CDCl₃): δ 12.55 (1H, s, OH-6), 7.49 (1H, d, J=6.9 Hz,H-10), 5.87 (1H, t, J=7.3 Hz, H-27), 5.00 (1H, br t, J=6.3 Hz, H-32),3.46 (1H, t, J=5.6 Hz, H-11), 3.27 (1H, dd, J=14.3, 8.6 Hz, H₁-31), 3.17(1H, dd, J=15.9, 8.4 Hz, H₁-26), 3.10 (1H, br dd, J=13.9, 4.4 Hz,H₂-31), 2.93 (1H, m, H-3), 2.92 (1H, m, H₂-26), 2.49 (1H, d, J=9.3 Hz,H-22), 2.40 (1H, t, J=8.4 Hz, H-4), 2.30 (1H, m, H-37), 2.28 (1H, m,H₁-21), 1.72 (1H, m, H₁-20), 1.70 (3H, s, H-34), 1.68 (3H, s, H-25),1.66 (3H, s, H-29), 1.60 (3H, s, H-35), 1.58 (1H, m, H₁-36), 1.52 (1H,m, H₂-20), 1.50 (1H, m, H₂-36), 1.35 (1H, dd, J=13.3, 9.6 Hz, H₂-21),1.28 (3H, s, H-19), 1.27 (6H, s, H-24, H-40), 0.69 (3H, s, H-39).

¹³C-NMR (150 MHz, CDCl₃): δ 203.75 (C-12), 178.86 (C-8), 171.59 (C-30),161.51 (C-18), 161.15 (C-6), 155.01 (C-16), 138.12 (C-27), 134.58(C-10), 133.85 (C-9), 130.79 (C-33), 128.07 (C-28), 122.35 (C-32),108.85 (C-17), 105.28 (C-5), 100.34 (C-7), 90.32 (C-14), 85.12 (C-2),84.20 (C-23), 83.80 (C-13), 48.93 (C-22), 46.79 (C-11), 46.19 (C-37),38.77 (C-38), 38.53 (C-20), 36.90 (C-4), 34.97 (C-3), 33.41 (C-40),29.90 (C-25), 29.19 (C-26), 28.91 (C-24), 27.31 (C-19), 25.73 (C-35),25.59 (C-36), 25.29 (C-21), 21.80 (C-31), 20.77 (C-29), 18.16 (C-34),17.65 (C-39).

The EIMS data of Product Gh-2607-1A show a molecular ion peak [M]⁺ atm/z 628 and the HREIMS data of Product Gh-2607-1A show a molecular ionpeak [M]⁺ at m/z 628.3046, indicating that Product Gh-2607-1A has amolecular formula identical to those of the above-described compoundsgambogic acid and gambogellic acid as well as their epimers, i.e.,C₃₈H₄₄O₈.

The ¹H-NMR and ¹³C-NMR data of Product Gh-2607-1A show that ProductGh-2607-1A does not have a cis disubstituted double bond in the pyranring thereof, nor does it have an isopropenyl group having a terminaldouble bond.

As further confirmed by the ¹H-¹H COSY, HMQC and HMBC analyses, ProductGh-2607-1A is structurally similar to gambogellic acid except for at themonoterpene moiety. As compared to gambogellic acid, Product Gh-2607-1Awas found to have one less double bond. Product Gh-2607-1A was thereforepresumed to have one more ring than gambogellic acid.

The ¹H-¹H COSY data of product Gh-2607-1A show that: a methine proton (δ2.93 (1H, m, H-3)) is coupled to another methine proton (δ 2.40 (1H, t,J=8.4 Hz, H-4)); the methine proton δ 2.40 is coupled to a furthermethine proton (δ 2.30 (1H, m, H-37)); the methine proton δ 2.30 iscoupled to one methylene proton (δ 1.50 (1H, m, H₂-36)) of C-36; and theother methylene proton (δ 1.58 (1H, m, H₁-36)) of C-36 is coupled toanother methylene proton (δ 1.72 (1H, m, H₁-20)), evidencing that themonoterpene moiety of Product Gh-2607-1A has two adjacent methylenegroups (C-20 (δ 38.53) and C-36 (δ 25.59)) and three adjacent methinegroups (C-37 (δ 46.19), C-4 (δ 36.90) and C-3 (δ 34.97)). The methinegroup (C-3) of Product Gh-2607-1A is a transformation of the methylenegroup (C-3) of gambogellic acid, and said transformation is presumed tobe caused by forming a bond between C-3 and C-38 to replace a terminaldouble bond between C-38 and C-40, thereby resulting in the formation ofa pinane-type monoterpene structure, which includes a cyclobutane, agem-dimethyl group (C-39 (δ 17.65) and C-40 (δ 33.41)), anoxygen-bearing quaternary carbon (C-2 (δ 85.12)), and a tertiary methylgroup (C-19 (δ 27.31)) attached to the oxygen-bearing quaternary carbon.

The HMBC data of Product Gh-2607-1A show that: in addition to beingcorrelated with aromatic ring carbons δ 105.28 (C-5), δ 161.15 (C-6) andδ 161.51 (C-18), δ 2.93 (H-3) is correlated with δ 85.12 (C-2), δ 46.19(C-37), δ 38.77 (C-38), δ 38.53 (C-20), δ 36.90 (C-4), δ 33.41 (C-40)and δ 17.65 (C-39); δ 2.40 (H-4) is correlated with δ 105.28 (C-5), δ38.77 (C-38), δ 34.97 (C-3), δ 46.19 (C-37), δ 25.59 (C-36), δ 38.53(C-20) and δ 27.31 (C-19); and δ 2.30 (H-37) is correlated with δ 105.28(C-5), δ 85.12 (C-2), δ 38.77 (C-38), δ 34.97 (C-3), δ 36.90 (C-4), δ38.53 (C-20), δ 25.59 (C-36) and δ 33.41 (C-40). It can be known fromthe above HMBC data that in the pinane-type monoterpene structure ofProduct Gh-2607-1A, C-2 is connected to C-18 of an aromatic ring via anether bond; C-4 is connected to C-5; and the methyl group (C-19)attached to C-2 is in an equatorial orientation.

The NOESY data of Product Gh-2607-1A show that: δ 7.49 (H-10) iscorrelated with δ 3.46 (H-11); δ 3.46 (H-11) is correlated with δ 2.28(H₁-21); δ 1.35 (H₂-21) is correlated with δ 2.49 (H-22); δ 2.49 (H-22)is correlated with δ 1.68 (H-25); and δ 5.87 (H-27) is correlated with δ1.66 (H-29), evidencing that the stereostructure of Product Gh-2607-1Ain this part has a 11S,13R,14S,22S configuration, which includes H-27and a carboxyl group (C-30) in a trans-relationship, and a double bondΔ^(27,28) in a Z configuration. In addition, δ 2.93 (H-3) is correlatedwith δ 2.40 (H-4), δ 1.28 (H-19) and δ 1.27 (H-40); δ 2.40 (H-4) iscorrelated with δ 2.30 (H-37), δ 1.27 (H-40) and δ 1.66 (H-29); δ 2.30(H-37) is correlated with δ 1.66 (H-29) and δ 1.70 (H-34); and δ 1.50(H₂-36) is correlated with δ 1.66 (H-29). It can therefore be known thatC-2 is in a S configuration; and in the pinane structure, the threemethine protons H-3, H-4 and H-37 in the cyclobutane ring are incis-relationship such that the cyclobutane ring is in a 3S,4S,37Rconfiguration. The NOESY data also reveal that: both δ 3.27 (H₁-31) andδ 3.10 (H₂-31) are correlated with δ 1.66 (H-29); both δ 3.17 (H₁-26)and δ 2.92 (H₂-26) are correlated with δ 1.70 (H-34); and δ 0.69 (H-39)is correlated with δ 1.72 (H₁-20), δ 1.28 (H-19) and δ 1.27 (H-40).

Based on the above information, Product Gh-2607-1A is identified to be anew compound having the following chemical structure:

Product Gh-2607-1A is identified by the name “epiformoxanthone B” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,9R,10R,12S,15S,18aS)-3a,4,5,7,12,13,14,15-octahydro-8-hydroxy-3,3,11,11,15-pentamethyl-17-(3-methyl-2-butenyl)-7,19-dioxo-1,5-methano-1H,3H,9H-furo[3.4-g]-16-oxa-tricyclo[4.4.0.0^(9,12)]decano[3.2-b]xanthen-1-yl]-(2Z)-]}.

15. Product Gh-2508:

Product Gh-2508, which was purified from eluate 25 of fraction 3, wasdetermined to have the following properties:

Yellow powder; mp 113˜118° C.

EIMS m/z (relative intensity): 628 [M]⁺ (71), 600 (100), 575 (14), 545(52), 517 (68), 501 (12), 473 (96), 459 (12), 431 (26), 417 (18), 389(29), 355 (64), 349 (26), 307 (12), 295 (22), 253 (24), 245 (29), 214.9(26), 189 (17), 105 (15), 91 (18), 69 (35).

¹H-NMR (600 MHz, CDCl₃): δ 12.57 (1H, s, OH-6), 7.50 (1H, d, J=6.9 Hz,H-10), 5.79 (1H, dd, J=16.0, 7.2 Hz, H-27), 5.30 (1H, d, J=16.0 Hz,H-26), 5.14 (1H, br t, J=6.9 Hz, H-32), 4.57 (1H, s, H₁-40), 4.23 (1H,s, H₂-40), 3.50 (1H, br d, J=2.8 Hz, H-4), 3.43 (1H, dd, J=6.8, 4.4 Hz,H-11), 3.23 (2H, m, H-31), 2.90 (1H, dq, J=7.2, 7.1 Hz, H-28), 2.56 (1H,d, J=9.3 Hz, H-22), 2.29 (1H, dd, J=13.4, 4.6 Hz, H₁-21), 2.16 (1H, brd, J=12.5 Hz, H-37), 1.94 (1H, br d, J=12.9 Hz, H₁-20), 1.89 (1H, m,H₁-3), 1.86 (3H, s, H-39), 1.75 (1H, m, H₂-3), 1.73 (6H, s, H-25, H-34),1.65 (3H, s, H-35), 1.56 (1H, dt, J=13.4, 5.0 Hz, H₂-20), 1.42 (1H, m,H₂-21), 1.38 (3H, s, H-19), 1.36 (2H, m, H-36), 1.27 (3H, s, H-24), 0.90(3H, d, J=7.1 Hz, H-29).

¹³C-NMR (150 MHz, CDCl₃): δ 203.38 (C-12), 179.21 (C-8), 177.35 (C-30),164.29 (C-18), 160.61 (C-6), 155.79 (C-16), 147.66 (C-38), 134.54 (C-9,C-10), 134.33 (C-27), 131.17 (C-33), 123.80 (C-26), 122.45 (C-32),108.83 (C-40), 106.65 (C-17), 104.18 (C-5), 100.11 (C-7), 91.46 (C-14),84.76 (C-13), 84.13 (C-23), 77.07 (C-2), 48.27 (C-22), 48.13 (C-37),47.06 (C-11), 41.74 (C-28), 39.31 (C-20), 36.92 (C-3), 30.03 (C-25),28.94 (C-4), 28.73 (C-24), 28.50 (C-19), 25.74 (C-35), 25.51 (C-21),22.98 (C-39), 22.74 (C-36), 21.94 (C-31), 18.18 (C-34), 15.80 (C-29).

The EIMS data of Product Gh-2508 show a molecular ion peak [M]⁺ at m/z628, which reveals that the fragmentation pattern of Product Gh-2508 isidentical to that of Product Gh-2603-2 (i.e., gambogellic acid).Besides, the ¹H-NMR and ¹³C-NMR data of Product Gh-2508 are generallysimilar to those of Product Gh-2603-2.

The ¹H-NMR and ¹³C-NMR data of Product Gh-2508 show that the side chainconnected to C-13 and containing a carboxyl group is different from thatof Product Gh-2603-2; the disubstituted double bond Δ^(26,27) (δ 5.79(1H, dd, J=16.0, 7.2 Hz, H-27) and δ 134.33 (C-27), and δ 5.30 (1H, d,J=16.0 Hz, H-26) and δ 123.80 (C-26)) exists instead of thetrisubstituted double bond Δ^(27,28) and the methylene group (C-26) inProduct Gh-2603-2. Besides, since two coupled olefinic protons (H-26 andH-27) have a coupling constant (J) of 16.0 Hz, the double bond Δ^(26,27)is a trans double bond (namely, in an E-configuration). It can be knownfrom a doublet signal of δ 0.90 (3H, d, J=7.1 Hz, H-29) that C-29represents a secondary methyl group and H-29 is coupled to a methineproton (δ 2.90 (1H, dq, J=7.2, 7.1 Hz, H-28)). Besides, a methine group(δ 2.90 (H-28) and δ 41.74 (C-28)) is adjacent to a carboxyl carbon (δ177.35 (C-30)), indicating that the carboxyl carbon is not anα/β-unsaturated carbonyl carbon.

The HMBC data of Product Gh-2508 show that: δ 5.79 (H-27) is correlatedwith δ 177.35 (C-30), δ 15.80 (C-29), δ 41.74 (C-28) and δ 84.76 (C-13);δ 5.30 (H-26) is correlated with δ 177.35 (C-30), δ 41.74 (C-28), δ134.33 (C-27) and δ 84.76 (C-13); δ 2.90 (H-28) is correlated with δ177.35 (C-30), δ 15.80 (C-29), δ 134.33 (C-27) and δ 123.80 (C-26); andδ 0.90 (H-29) is correlated with δ 177.35 (C-30) and δ 134.33 (C-27),evidencing that the side chain connected to C-13 is(E)-2-methyl-3-butenoic acid.

The NOESY data of Product Gh-2508 show that δ 7.50 (H-10) is correlatedwith δ 3.43 (H-11); δ 3.43 (H-11) is correlated with δ 2.29 (H₁-21); andδ 2.56 (H-22) is correlated with δ 1.42 (H₂-21) and δ 1.27 (H-24),evidencing that the stereostructure of Product Gh-2508 in this part isidentical to that of gambogic acid or gambogellic acid, i.e., in a11S,13R,14S,22S configuration. Besides, δ 0.90 (H-29) is correlated withnot only δ 5.79 (H-27) and δ 5.30 (H-26) but also the adjacent δ 2.90(H-28).

As further confirmed by the ¹H-¹H COSY, HMQC and HMBC analyses, themonoterpene moiety of Product Gh-2508 is identical to that of ProductGh-2603-2. The signal of the methine proton (δ 2.16 (1H, br d, J=12.5Hz, H-37)) attached to the isopropenyl group reveals that the methineproton attached to the isopropenyl group and a vicinal proton have arelatively high vicinal coupling constant (J=12.5 Hz) and, hence, theyare in an axial-axial coupling relationship. As such, H-37 is in anaxial orientation, and C-37 is in an S-configuration.

The NOESY data of Product Gh-2508 show that: δ 3.50 (H-4) is correlatedwith not only δ 2.16 (H-37) but also δ 1.89 (H₁-3) and δ 1.75 (H₂-3); δ1.89 (H₁-3) is correlated with δ 1.38 (H-19); and an axial hydrogen δ2.16 (H-37) is correlated with δ 1.89 (H₁-3) and δ 1.56 (H₂-20),evidencing that the monoterpene ring has a chair conformation with1,3-diaxial interaction, and both the methyl proton (H-19) connected toC-2 and the isopropenyl group connected to C-37 are in an equatorialorientation. Inasmuch as no nuclear Overhauser effect (NOE) is presentamongst the methyl proton (δ 0.90 (H-29)) on the side chain attached toC-13 and the exo-methylene protons (δ 4.57 (H₁-40) and δ 4.23 (H₂-40))of the isopropenyl group in the monoterpene moiety, C-2 is presumed tobe in an R configuration. As such, the monoterpene moiety is determinedto be in a 2R,4R,37S configuration.

Based on the above information, Product Gh-2508 is identified to be anew compound having the following chemical structure:

Product Gh-2508 is identified by the name “β-gambogellic acid” {IUPACnomenclature: [3-butenoic acid,2-methyl-4-[(1R,3aS,5S,9R,10S,13R,16aS)-3a,4,5,7,10,11,12,13-octahydro-8-hydroxy-3,3,13-trimethyl-15-(3-methyl-2-butenyl)-10-(1-methylethenyl)-7,18-dioxo-1,5:9,13-dimethano-1H,3H,9H-furo[3.4-g]oxocino[3.2-b]xanthen-1-yl]-(3E)-]}.

16. Product Gh-2507:

Product Gh-2507, which was purified from eluate 23 of fraction 2, wasdetermined to have the following properties:

Yellow needles; mp 148˜152° C.

EIMS m/z (relative intensity): 628 [M]⁺ (48), 600 (100), 585 (9), 545(41), 517 (69), 510 (12), 473 (93), 431 (24), 417 (18), 389 (28), 355(68), 347 (25), 307 (11), 299 (24), 295 (20), 253 (22), 245 (28), 214.9(22), 199 (18), 189 (15), 105 (12), 91 (17), 69 (25).

¹H-NMR (600 MHz, CDCl₃): δ 12.61 (1H, s, OH-6), 7.48 (1H, d, J=6.9 Hz,H-10), 6.06 (1H, dd, J=15.7, 7.2 Hz, H-27), 5.15 (1H, d, J=15.7 Hz,H-26), 5.11 (1H, br t, J=6.9 Hz, H-32), 4.56 (1H, s, H₁-40), 4.20 (1H,s, H₂-40), 3.54 (1H, br d, J=2.7 Hz, H-4), 3.44 (1H, dd, J=6.8, 4.5 Hz,H-11), 3.25 (1H, dd, J=14.4, 8.1 Hz, H₁-31), 3.17 (1H, dd, J=14.4, 5.6Hz, H₂-31), 2.90 (1H, dq, J=7.2, 7.1 Hz, H-28), 2.56 (1H, d, J=9.3 Hz,H-22), 2.30 (1H, dd, J=13.5, 4.7 Hz, H₁-21), 2.16 (1H, br d, J=12.4 Hz,H-37), 1.97 (1H, br d, J=12.8 Hz, H₁-20), 1.88 (1H, m, H₁-3), 1.87 (3H,s, H-39), 1.74 (1H, m, H₂-3), 1.72 (3H, s, H-25), 1.71 (3H, s, H-34),1.64 (3H, s, H-35), 1.55 (1H, dt, J=13.5, 4.9 Hz, H₂-20), 1.44 (1H, m,H₂-21), 1.43 (1H, m, H₁-36), 1.36 (3H, s, H-19), 1.32 (1H, m, H₂-36),1.27 (3H, s, H-24), 0.92 (3H, d, J=7.0 Hz, H-29).

¹³C-NMR (150 MHz, CDCl₃): δ 203.53 (C-12), 179.16 (C-8), 177.27 (C-30),164.37 (C-18), 160.73 (C-6), 156.10 (C-16), 148.04 (C-38), 134.80(C-10), 134.65 (C-27), 134.52 (C-9), 131.20 (C-33), 122.91 (C-26),122.39 (C-32), 108.71 (C-40), 106.53 (C-17), 104.14 (C-5), 99.93 (C-7),90.92 (C-14), 84.66 (C-13), 84.10 (C-23), 77.16 (C-2), 48.49 (C-22),48.15 (C-37), 47.03 (C-11), 42.00 (C-28), 39.21 (C-20), 37.04 (C-3),29.96 (C-25), 28.93 (C-4), 28.76 (C-24), 28.53 (C-19), 25.77 (C-35),25.35 (C-21), 22.34 (C-33), 22.53 (C-36), 22.01 (C-31), 18.15 (C-34),15.92 (C-29).

The EIMS data of Product Gh-2507 show a molecular ion peak [M]⁺ at m/z628, which is identical to those of Products Gh-2603-1 (i.e.,epigambogellic acid) and Gh-2603-2 (i.e., gambogellic acid). Besides,the ¹H-NMR and ¹³C-NMR data of Product Gh-2507 are generally similar tothose of Product Gh-2603-1.

The ¹H-NMR and ¹³C-NMR data of Product Gh-2507 show that a side chainconnected to C-13 and having a carboxyl group differs from that ofProduct Gh-2603-1; a disubstituted double bond Δ^(26,27) (δ 5.15 (1H, d,J=15.7 Hz, H-26) and δ 122.91 (C-26), and δ 6.06 (1H, dd, J=15.7, 7.2Hz, H-27) and δ 134.65 (C-27)] exists instead of the trisubstituteddouble bond Δ^(27,28) and the methylene group (C-26) in ProductGh-2603-1; and two coupled olefinic protons (H-26 and H-27) have acoupling constant (J) of 15.7 Hz. As such, the disubstituted double bondΔ^(26,27) is a trans double bond (namely, in an E-configuration). It canbe known from a doublet signal of δ 0.92 (3H, d, J=7.0 Hz, H-29) thatC-29 represents a secondary methyl group, and the proton (H-29) of thesecondary methyl group is coupled to a methine proton (δ 2.90 (1H, dq,J=7.2, 7.1 Hz, H-28)). In addition, since the methine group (δ 2.90(H-28) and δ 42.00 (C-28)) is adjacent to a carboxyl carbon (δ 177.27(C-30)), the carboxyl carbon is not an α,β-unsaturated carbonyl carbon.

The HMBC data of Product Gh-2507 show that: δ 6.06 (H-27) is correlatedwith δ 177.27 (C-30), δ 15.92 (C-29), δ 42.00 (C-28) and δ 84.66 (C-13);δ 5.15 (H-26) is correlated with δ 42.00 (C-28), δ 134.65 (C-27), δ84.66 (C-13) and δ 203.53 (C-12); δ 2.90 (H-28) is correlated with δ177.27 (C-30), δ 15.92 (C-29), δ 134.65 (C-27) and δ 122.91 (C-26); andδ 0.82 (H-29) is correlated with δ 177.27 (C-30), δ 42.00 (C-28) and δ134.65 (C-27), evidencing that the side chain attached to C-13 is(E)-2-methyl-3-butenoic acid.

The NOESY data of Product Gh-2507 show that: δ 7.48 (H-10) is correlatedwith δ 3.44 (H-11); δ 3.44 (H-11) is correlated with δ 2.30 (H₁-21) andδ 1.44 (H₂-21); δ 1.44 (H₂-21) is correlated with δ 2.56 (H-22); and δ2.56 (H-22) is correlated with δ 1.72 (H-25), evidencing that thestereostructure of Product Gh-2507 in this part is identical to that ofgambogic acid, gambogellic acid and epigambogellic acid, i.e., in a11S,13R,14S,22S configuration. Besides, δ 0.92 (H-29) is correlated withδ 2.90 (H-28), δ 6.06 (H-27), δ 5.15 (H-26), and δ 4.20 (H₂-40). Themethyl proton (H-29) is correlated with the exo-methylene protons (δ4.56 (H₁-40) and δ 4.20 (H₂-40)] of the isopropenyl group in themonoterpene moiety. As such, C-2 is presumed to be in a S-configuration.If C-2 is in a R configuration, no cross peaks would appear.

As further confirmed by the ¹H-¹H COSY, HMQC and HMBC analyses, themonoterpene moiety of Product Gh-2507 is identical to that of ProductGh-2603-1. The signal of the methine proton (δ 2.16 (1H, br d, J=12.4Hz, H-37)) attached to the isopropenyl group reveals that the methineproton attached to the isopropenyl group and a vicinal proton have arelatively high vicinal coupling constant (J=12.4 Hz) and, hence, theyare in an axial-axial coupling relationship. As such, H-37 is in anaxial orientation, and C-37 is in an R configuration.

The NOESY data of Product Gh-2507 also reveal that: δ 3.54 (H-4) iscorrelated with not only δ 2.16 (H-37) but also δ 1.88 (H₁-3) and δ 1.74(H₂-3): the methylene protons (H₁-3 and H₂-3) both are correlated with δ1.36 (H-19); and the axial hydrogen δ 2.16 (H-37) is correlated with notonly δ 1.87 (H-39) but also δ 1.88 (H₁-3), δ 1.55 (H₂-20) and δ 1.43(H₁-36), evidencing that the monoterpene ring has a chair conformationwith 1,3-diaxial interaction, in which both the methyl group (C-19)connected to C-2 and the isopropenyl group connected to C-37 are in anequatorial orientation. As such, the stereostructure of the p-menthenemonoterpene is in a 2S,4S,37R configuration.

Based on the above information, Product Gh-2507 is identified to be anew compound having the following chemical structure:

Product Gh-2507 is identified by the name “β-epigambogellic acid” {IUPACnomenclature: [3-butenoic acid,2-methyl-4-[(1R,3aS,5S,9S,10R,13S,16aS)-3a,4,5,7,10,11,12,13-octahydro-8-hydroxy-3,3,13-trimethyl-15-(3-methyl-2-butenyl)-10-(1-methylethenyl)-7,18-dioxo-1,5:9,13-dimethano-1H,3H,9H-furo[3.4-g]oxocino[3.2-b]xanthen-1-yl]-(3E)-]}.

17. Product Gh-2501:

Product Gh-2501, which was purified from eluate 21 of fraction 2, wasdetermined to have the following properties:

Yellow powder; mp 100˜103° C.

EIMS m/z (relative intensity): 544 [M]⁺ (100), 529 (67), 516 (16), 501(56), 473 (13), 435 (11), 405 (59), 389 (25), 363 (43), 349 (37), 337(12), 307 (34), 287 (58), 259 (29), 229 (69), 215 (59), 189 (35), 147(23), 135 (30), 105 (42), 91 (27), 83 (29), 69 (36), 55 (34).

¹H-NMR (600 MHz, CDCl₃): δ 12.70 (1H, s), 9.21 (1H, s), 7.54 (1H, d,J=7.0 Hz), 6.59 (1H, d, J=10.0 Hz), 6.37 (1H, t, J=8.5 Hz), 5.50 (1H, d,J=10.0 Hz), 5.07 (1H, t, J=8.4 Hz), 3.51 (1H, dd, J=6.9, 4.5 Hz), 3.25(1H, dd, J=14.4, 8.1 Hz), 3.17 (1H, br dd, J=13.9, 5.9 Hz), 2.71 (1H,ddd, J=16.0, 7.5, 0.8 Hz), 2.62 (1H, ddd, J=16.0, 7.0, 0.9 Hz), 2.56(1H, d, J=9.4 Hz), 2.34 (1H, dd, J=13.6, 4.7 Hz), 1.73 (3H, s), 1.71(3H, s), 1.62 (3H, s), 1.43 (3H, s), 1.41 (3H, s), 1.39 (1H, m), 1.29(3H, s), 1.28 (3H, d, J=1.2 Hz).

¹³C-NMR (150 MHz, CDCl₃): δ 203.02, 194.46, 178.84, 161.34, 157.68,157.15, 146.51, 140.11, 135.62, 133.34, 131.98, 126.39, 121.82, 115.28,108.07, 103.27, 100.36, 90.79, 83.98, 83.39, 78.87, 48.99, 46.85, 29.96,28.96, 28.93, 28.39 (2C), 25.75, 25.26, 21.67, 18.16, 8.58.

According to the obtained spectral data, Product Gh-2501 was identifiedto be a known compound having the following chemical structure, i.e.,isomorellin:

18. Product Gh-2505:

Product Gh-2505, which was purified from eluate 24 of fraction 2, wasdetermined to have the following properties:

Yellow powder; mp 67˜70° C.

EIMS m/z (relative intensity): 630 [M]⁺ (100), 602 (19), 545 (14), 507(36), 479 (22), 475 (18), 433 (8), 351 (27), 309 (17), 295 (38), 253(45), 245 (16), 231 (16), 213 (13), 177 (15), 147 (8), 69 (29).

¹H-NMR (600 MHz, CDCl₃): δ 12.81 (1H, s), 7.52 (1H, d, J=6.9 Hz), 6.48(1H, s), 5.83 (1H, t, J=7.2 Hz), 5.17 (1H, t, J=6.9 Hz), 5.06 (1H, brt,J=6.6 Hz), 5.02 (1H brt, J=6.7 Hz), 3.48 (1H, dd, J=6.6, 4.8 Hz), 3.30(1H, m), 3.28 (2H, m), 3.24 (1H, dd, J=16.1, 7.1 Hz), 3.10 (1H, dd,J=15.7, 8.7 Hz), 2.86 (1H, ddd, J=15.9, 6.7, 1.1 Hz), 2.49 (1H, d, J=9.4Hz), 2.30 (1H, dd, J=13.5, 4.7 Hz), 2.06 (2H, m), 2.00 (2H, m), 1.74(3H, s), 1.71 (3H, s), 1.70 (3H, s), 1.66 (3H, s), 1.65 (3H, s), 1.64(3H, s), 1.56 (3H, s), 1.36 (1H, dd, J=13.5, 9.5 Hz), 1.27 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 203.38, 179.12, 170.39, 163.62, 160.34,155.85, 139.06, 136.95, 135.15, 133.91, 133.52, 131.88, 128.50, 123.83,121.91, 121.35, 107.50, 106.43, 100.64, 90.41, 83.94, 83.87, 48.90,46.88, 39.68, 29.79, 29.46, 28.92, 26.33, 25.71, 25.67, 25.21, 22.03,21.09, 20.74, 17.98, 17.68, 16.16.

According to the obtained spectral data, Product Gh-2505 was identifiedto be a known compound having the following chemical structure, i.e.,gambogenic acid:

19. Product Gh-2642:

Product Gh-2642, which was purified from eluate 22 of fraction 2, wasdetermined to have the following properties:

Yellow powder; mp 157˜159° C.

EIMS m/z (relative intensity): 630 [M]⁺ (100), 602 (11), 545 (11), 533(16), 507 (46), 479 (21), 475 (15), 433 (7), 419 (8), 381 (9), 357 (13),351 (20), 309 (14), 295 (27), 253 (34), 245 (15), 231 (16), 213 (0.11),177 (15), 147 (8), 135 (8), 105 (11), 69 (44).

¹H-NMR (600 MHz, CDCl₃): δ 12.76 (1H, s), 7.52 (1H, d, J=7.0 Hz), 6.70(1H, br s), 6.59 (1H, t, J=7.5 Hz), 5.19 (1H, t, J=7.0 Hz), 5.10 (1H, brt, J=6.0 Hz), 5.02 (1H, br t, J=6.1 Hz), 3.47 (1H, t, J=5.6 Hz), 3.34(2H, m), 3.31 (2H, m), 2.60 (1H, dd, J=15.7, 7.9 Hz), 2.50 (1H, m), 2.49(1H, d, J=9.5 Hz), 2.30 (1H, dd, J=13.3, 4.4 Hz), 2.06 (2H, m), 2.02(2H, m), 1.76 (3H, s), 1.70 (3H, s), 1.66 (6H, s), 1.62 (3H, s), 1.54(3H, s), 1.35 (1H, dd, J=13.3, 9.5 Hz), 1.30 (3H, s), 1.25 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 203.15, 179.02, 172.20, 163.51, 160.27,155.87, 138.98, 136.90, 135.50, 133.72, 133.33, 131.75, 128.64, 123.81,121.88, 121.15, 107.51, 106.63, 100.55, 90.37, 83.65, 83.49, 48.89,46.82, 39.61, 29.80, 28.85 (2C), 26.27, 25.65, 25.60, 25.19, 21.98,21.10, 17.93, 17.59, 16.16, 11.34.

According to the obtained spectral data, Product Gh-2642 was identifiedto be a known compound having the following chemical structure, i.e.,isogambogenic acid:

20. Product Gh-1601-A:

Product Gh-1601-A, which was purified from eluate 20 of fraction 2, wasdetermined to have the following properties:

Yellow powder; mp 143˜145° C.

EIMS m/z (relative intensity): 644 [M]⁺ (72), 598 (18), 561 (100), 515(23), 474 (35), 431 (9), 391 (10), 355 (14), 349 (7), 347 (6), 253 (6),248 (11), 215 (11), 189 (6), 125 (4), 69 (18).

¹H-NMR (600 MHz, CDCl₃): δ 12.76 (1H, s), 7.54 (1H, d, J=6.9 Hz), 6.61(1H, d, J=10.2 Hz), 6.31 (1H, t, J=7.2 Hz), 5.41 (1H, d, J=10.1 Hz),5.02 (2H, br s), 4.09 (1H, d, J=13.3 Hz), 4.01 (1H, d, J=13.3 Hz), 3.49(1H, br t, J=5.6 Hz), 3.27 (1H, dd, J=14.2, 8.0 Hz), 3.13 (1H, br dd,J=14.3, 5.2 Hz), 2.96 (2H, d, J=7.5 Hz), 2.51 (1H, d, J=9.6 Hz), 2.31(1H, dd, J=13.4, 4.7 Hz), 2.00 (2H, m), 1.72 (1H, m), 1.70 (3H, s), 1.67(3H, s), 1.62 (3H, s), 1.60 (3H, s), 1.58 (1H, m), 1.52 (3H, s), 1.38(1H, m), 1.37 (3H, s), 1.26 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 203.04, 179.01, 169.43, 161.66, 157.50,157.27, 139.33, 135.66, 133.20, 131.90, 131.87, 131.24, 124.80, 123.69,121.96, 115.76, 107.75, 102.82, 100.47, 90.76, 84.25, 83.67, 81.47,64.80, 48.92, 46.82, 41.93, 29.82, 29.14, 28.82, 27.76, 25.68, 25.65,25.12, 22.70, 21.60, 18.13, 17.61.

According to the obtained spectral data, Product Gh-1601-A wasidentified to be a known compound having the following chemicalstructure, i.e., 30-hydroxygambogic acid:

21. Product Gh-1602:

Product Gh-1602, which was purified from eluate 18 of fraction 2, wasdetermined to have the following properties:

Yellow powder; mp 98˜100° C.

EIMS m/z (relative intensity): 644 [M]⁺ (28), 598 (5), 561 (100), 515(15), 474 (9), 431 (3), 389 (5), 355 (5), 347 (5), 253 (3), 245 (6), 215(10), 189 (5), 125 (3), 69 (8).

¹H-NMR (600 MHz, CDCl₃): δ 12.73 (1H, s), 7.54 (1H, d, 0.7=6.9 Hz), 6.60(1H, d, J=10.1 Hz), 6.40 (1H, t, J=7.4 Hz), 5.39 (1H, d, J=10.2 Hz),5.07 (1H, t, J=7.0 Hz), 5.01 (1H, brt, J=6.8 Hz), 4.09 (1H, d, J=13.1Hz), 4.02 (1H, d, J=13.2 Hz), 3.46 (1H, t, J=5.6 Hz), 3.27 (1H, dd,J=14.6, 8.2 Hz), 3.12 (1H, dd, J=14.6, 5.0 Hz), 2.96 (2H, d, J=7.5 Hz),2.50 (1H, d, J=9.3 Hz), 2.29 (1H, dd, J=13.5, 4.7 Hz), 2.04 (2H, m),1.73 (1H, m), 1.71 (3H, s), 1.67 (3H, s), 1.64 (3H, s), 1.62 (1H, m),1.61 (3H, s), 1.56 (3H, s), 1.36 (1H, m), 1.32 (3H, s), 1.26 (3H, s).

¹³C-NMR (150 MHz, CDCl₃): δ 203.08, 179.02, 169.57, 161.46, 157.50,157.24, 140.20, 135.91, 133.02, 132.20, 131.62, 131.15, 124.94, 123.74,122.04, 115.81, 107.89, 102.95, 100.50, 90.89, 84.02, 83.56, 81.21,64.64, 48.91, 46.82, 41.67, 29.90, 29.14, 28.77, 26.91, 25.69, 25.61,25.19, 22.73, 21.58, 18.15, 17.60.

According to the obtained spectral data, Product Gh-1602 was identifiedto be a known compound having the following chemical structure, i.e.,30-hydroxyepigambogic acid:

22. Product Gh-2641-1:

Product Gh-2641-1, which was purified from eluate 20 of fraction 2, wasdetermined to have the following properties:

Yellow powder; mp 94˜98° C.

EIMS m/z (relative intensity): 646 [M]⁺ (100), 545 (18), 523 (80), 495(28), 477 (17), 449 (16), 367 (44), 349 (20), 325 (37), 295 (41), 252.9(49), 245 (27), 213 (32), 147 (16).

¹H-NMR (600 MHz, CDCl₃): δ 12.84 (1H, s, OH-6), 7.46 (1H, d, J=7.0 Hz,H-10), 5.30 (1H, ddd, J=12.1, 4.0, 1.4 Hz, H-27), 5.19 (1H, dt, J=7.1,1.2 Hz, H-32), 5.04 (1H, tt, J=6.9, 1.3 Hz, H-37), 4.68 (1H, dd, J=9.8,6.5 Hz, H-4), 3.76 (1H, dd, J=14.5, 12.1 Hz, H₁-26), 3.48 (1H, d, J=7.0,4.2 Hz, H-11), 3.16 (2H, m, H-31), 3.13 (1H, dd, J=15.3, 6.5 Hz, H₁-3),3.03 (1H, dd, J=15.3, 9.8 Hz, H₂-3), 2.76 (1H, ddd, J=14.5, 4.0, 2.2 Hz,H₂-26), 2.39 (1H, d, J=9.6 Hz, H-22), 2.32 (1H, dd, J=13.5, 4.5 Hz,H₁-21), 2.02 (2H, m, H-36), 1.91 (2H, m, H-20), 1.70 (3H, s, H-35), 1.64(3H, s, H-25), 1.61 (3H, s, H-39), 1.53 (3H, s, H-40), 1.51 (3H, s,H-29), 1.43 (3H, s, H-19), 1.33 (1H, m, H₂-21), 1.28 (3H, s, H-24), 1.25(3H, s, H-34).

¹³C-NMR (150 MHz, CDCl₃): δ 202.10 (C-12), 178.09 (C-8), 168.66 (C-30),167.88 (C-18), 163.12 (C-16), 152.96 (C-6), 135.41 (C-33), 135.12(C-27), 134.13 (C-10), 133.72 (C-9), 131.18 (C-38), 129.76 (C-28),124.36 (C-37), 121.53 (C-32), 104.85 (C-5), 103.47 (C-17), 100.53 (C-7),90.43 (C-4), 90.21 (C-14), 84.37 (C-13), 83.66 (C-23), 73.26 (C-2),48.88 (C-22), 46.61 (C-11), 39.67 (C-20), 30.13 (C-26), 29.92 (C-25),29.15 (C-24), 26.65 (C-36, C-3), 26.60 (C-19), 25.60 (C-39), 25.19(C-21), 24.65 (C-35), 21.25 (C-31), 20.47 (C-29), 17.58 (C-40), 16.04(C-34).

The EIMS data of Product Gh-2641-1 show a molecular ion peak [M]⁺ at m/z646, which corresponds to xanthones having a molecular formula ofC₃₈H₄₅O₉.

The ¹H-NMR data of product Gh-2641-1 show that Product Gh-2641-1 has achelated hydroxy group (δ 12.84), four olefinic protons (δ 7.46, δ 5.30,δ 5.19, and δ 5.04), and a secondary hydroxy group (δ 4.68). As comparedto gambogic acid, the ¹H-NMR spectra of product Gh-2641-1 has one lesssignal of a coupled cis-disubstituted double bond and one more protonsignal of AX₂ spin system (δ 4.68 (1H, dd, J=9.8, 6.5 Hz, H-4), δ 3.13(1H, dd, J=15.3, 6.5 Hz, H₁-3), and δ 3.03 (1H, dd, J=15.3, 9.8 Hz,H₂-3)), which indicates that Product Gh-2641-1 may be a known compoundneogambogic acid.

The HMBC data of Product Gh-2641-1 show that: in addition to beingcorrelated with two quaternary carbons (δ 103.47 (C-17) and δ 167.88(C-18)), the hydroxymethine proton (δ 4.68 (1H, dd, J=9.8, 6.5 Hz, H-4))is correlated with an oxygen-bearing quaternary carbon (δ 73.26 (C-2))of a pyran ring and a tertiary methyl group (δ 26.60 (C-19)), evidencingthat the hydroxyl group is attached to C-4. It can be known from thecoupling constant of H-4 (J=9.8, 6.5 Hz) that the quasi-axial proton onC-4 is coupled to the quasi-axial H₂-3 (J=9.8 Hz) and thequasi-equatorial H₁-3 (J=6.5 Hz), respectively. As such, the hydroxygroup is in a β-orientation.

The NOESY data of Product Gh-2641-1 show that: δ 7.46 (H-10) iscorrelated with δ 3.48 (H-11); δ 3.48 (H-11) is correlated with δ 2.32(H₁-21); δ 2.32 (H₁-21) is correlated with δ 2.39 (H-22); δ 2.39 (H-22)is correlated with δ 1.28 (H-24); and δ 5.30 (H-27) is correlated with δ1.51 (H-29), evidencing that the stereo structure of product Gh-2641-1in this part is identical to that of gambogic acid, i.e., in a11S,13R,14S,22S configuration, which includes H-27 and carboxyl group(C-30) in a trans relationship, and a double bond Δ^(27,28) in a Zconfiguration. In addition, δ 4.68 (H-4) is correlated with δ 1.43(H-19), which indicates that the methyl proton connected to C-2 is in anaxial orientation (or α-orientation). As such, C-2 of Product Gh-2641-1has a configuration identical to that of gambogic acid, i.e., in an Rconfiguration.

According to the obtained spectral data, Product Gh-2641-1 is identifiedto be a known compound having the following chemical structure, i.e.,neogambogic acid:

23. Product Gh-1631:

Product Gh-1631, which was purified from eluate 19 of fraction 2, wasdetermined to have the following properties:

Yellow needles; mp 95˜97° C.

EIMS m/z (relative intensity): 646 [M]⁺ (54), 618 (75), 573 (12), 545(8), 520 (13), 492 (75), 491 (100), 477 (18), 449 (38), 373 (19), 349(11), 321 (15), 295 (27), 267 (10), 252.9 (37), 245 (18), 213 (11),188.9 (15), 176.9 (11), 109 (24), 99 (20), 69 (83); HREIMS [M]⁺ m/z:646.3146; calculated for C₃₈H₄₆O₉, 646.3142.

¹H-NMR (600 MHz, CDCl₃): δ 12.45 (1H, s, OH-6), 7.42 (1H, d, J=6.9 Hz,H-10), 5.32 (1H, br d, J=9.9 Hz, H-27), 5.18 (1H, t, J=6.8 Hz, H-32),5.08 (1H, t, J=7.1 Hz, H-37), 4.75 (1H, t, J=8.1 Hz, H-3), 3.53 (1H, dd,J=15.7, 11.0 Hz, H₁-26), 3.48 (1H, dd, J=6.6, 4.8 Hz, H-11), 3.29 (1H,dd, J=15.1, 7.3 Hz, H₁-31), 3.24 (1H, dd, J=15.2, 6.3 Hz, H₂-31), 3.07(2H, d, J=8.1 Hz, H-4), 2.69 (1H, ddd, J=15.8, 4.0, 2.3 Hz, H₂-26), 2.53(1H, d, J=9.4 Hz, H-22), 2.30 (1H, dd, J=13.5, 4.8 Hz, H₁-21), 2.09 (1H,m, H₁-36), 2.02 (1H, m, H₂-36), 1.72 (3H, s, H-34), 1.67 (3H, s, H-35),1.66 (3H, s, H-39), 1.65 (3H, s, H-25), 1.601 (3H, s, H-40), 1.597 (3H,sh, H-29), 1.57 (1H, m, H₁-20), 1.47 (1H, m, H₂-20), 1.42 (1H, m,H₂-21), 1.42 (3H, s, H-19), 1.23 (3H, s, H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 204.15 (C-12), 179.64 (C-8), 168.08 (C-30),168.02 (C-18), 158.45 (C-16), 157.32 (C-6), 136.74 (C-27, C-33), 134.37(C-10), 132.40 (C-38), 132.12 (C-9), 128.40 (C-28), 123.56 (C-37),121.83 (C-32), 105.90 (C-5), 103.96 (C-17), 101.72 (C-7), 90.28 (C-14),90.00 (C-3), 84.40 (C-13), 83.45 (C-23), 75.77 (C-2), 75.70 (C-2), 48.78(C-22), 47.71 (C-11), 36.93 (C-20), 29.87 (C-25), 29.32 (C-26), 28.87(C-24), 25.93 (C-4), 25.71 (C-35), 25.64 (C-39), 25.14 (C-21), 23.54(C-19), 22.51 (C-31), 22.06 (C-36), 20.91 (C-29), 17.94 (C-34), 17.66(C-40).

The EIMS data of Product Gh-1631 show a molecular ion peak [M]⁺ at m/z646 (54) and a base peak at m/z 491 (100), and the HREIMS data ofProduct Gh-1631 show [M]⁺ at m/z 646.3146, indicating that ProductGh-1631 has a molecular formula identical to that of Product Gh-2641-1(i.e., neogambogic acid), namely, C₃₈H₄₆O₉.

The ¹H-NMR data of Product Gh-1631 show that: Product Gh-1631 has achelated hydroxy group (δ 12.45), four olefinic protons (δ 7.42, δ 5.32,δ 5.18, and δ 5.08), and a secondary hydroxy group having ahydroxymethine proton (δ 4.75 (1H, t, J=8.1 Hz)) coupled with amethylene proton (δ 3.07 (2H, d, J=8.1 Hz)). The ¹H-NMR data of ProductGh-1631 are generally similar to those of Product Gh-2641-1.

The HMQC data of Product Gh-1831 reveal that: δ 90.00 (—OCH—) and δ25.93 (—CH₂—) are signals that correspond to a hydroxymethine carbon anda methylene carbon adjacent to the hydroxymethine carbon, respectively.As such, it is presumed that Product Gh-1631 and Product Gh-2641-1 areisomers that differ from each other at the site of the hydroxy group,and Product Gh-1631 may have the hydroxy group connected to C-3.

The HMBC data of Product Gh-1631 show that: δ 4.75 is correlated with δ168.02 (C-18), δ 105.90 (C-5), δ 75.77 (C-2), δ 75.70 (C-2), δ 25.93(C-4) and δ 23.54 (C-19); and δ 3.07 is correlated with δ 90.00 (C-3), δ105.90 (C-5), δ 75.77 (C-2), δ 75.70 (C-2), δ 157.32 (C-6), δ 168.02(C-18), δ 103.96 (C-17), δ 101.72 (C-7) and δ 158.45 (C-16), evidencingthat the hydroxyl group is connected to C-3. In addition, since noconjugated double bond is present therein, the conformation of the pyranring is in a flexible form, which renders the two protons of the C-4methylene group to be in equivalence and have identical chemical shift(δ 3.07 (2H, d, J=8.1 Hz, H-4)), in which δ 3.07 is correlated with δ4.75 (1H, t, J=8.1 Hz, H-3). As such, the hydroxy group on C-3 is in anaxial orientation (or β-orientation), and C-3 is in an R configuration.

The NOESY data of Product Gh-1631 reveal that: δ 7.42 (H-10) iscorrelated with δ 3.48 (H-11); δ 3.48 (H-11) is correlated with δ 2.30(H₁-21); δ 2.53 (H-22) is correlated with δ 1.42 (H₂-21); δ 2.30 (H₁-21)is correlated with δ 1.23 (H-24); and δ 5.32 (H-27) is correlated with δ1.597 (H-29), evidencing that the stereostructure of product Gh-1631 inthis part is identical to that of gambogic acid, i.e., in a11S,13R,14S,22S configuration, which includes H-27 and carboxyl group(C-30) in a trans relationship, and a double bond Δ^(27,28) in a Zconfiguration. In addition, δ 4.75 (H-3) is correlated with δ 3.07(H-4), δ 1.42 (H-10), δ 1.47 (H₂-20) and δ 2.02 (H₂-36), which indicatesthat the C-3 proton is in an equatorial orientation (α-orientation), andthe methyl group attached to C-2 is in an axial orientation(α-orientation). As such, C-2 is in an R configuration.

Based on the above information, Product Gh-1631 is identified to be anew compound having the following chemical structure:

Product Gh-1631 is identified by the name “formoxanthone C” {IUPACnomenclature [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,10R,11R,14aS)-3a,4,5,7,10,11-hexahydro-8,10-dihydroxy-3,3,11-trimethyl-13-(3-methyl-2-butenyl)-11-(4-methyl-3-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,9H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

24. Product Gh-1050:

Product Gh-1050, which was purified from eluate 17 of fraction 2, wasdetermined to have the following properties:

Yellow powder; mp 55˜57° C.

EIMS m/z (relative intensity): 644 [M]⁺ (82), 616 (98), 601 (9), 598(8), 571 (16), 533 (17), 517 (12), 490 (100), 489 (96), 475 (19), 447(30), 433 (13), 405 (18), 371 (33), 363 (17), 309 (10), 295 (21), 253(25), 230 (19), 213 (15), 189 (9), 173 (10), 147 (11), 105 (17), 99(25), 69 (42); HREIMS [M]⁺ m/z: 644.2983; calculated for C₃₈H₄₄O₉,644.2985.

¹H-NMR (600 MHz, CDCl₃): δ 12.69 (1H, s, OH-6), 7.47 (1H, d, J=6.8 Hz,H-10), 5.57 (1H, t, J=7.5 Hz, H-27), 5.19 (1H, t, J=6.8 Hz, H-32), 4.63(1H, s, H₁-40), 4.29 (1H, s, H₂-40), 3.86 (1H, d, J=3.4 Hz, H-3), 3.65(1H, t, J=2.6 Hz, H-4), 3.49 (1H, t, J=5.7 Hz, H-11), 3.31 (1H, m,H₁-31), 3.28 (1H, m, H₁-26), 3.23 (1H, dd, J=14.6, 6.2 Hz, H₂-31), 2.82(1H, dd, J=14.9, 5.6 Hz, H₂-26), 2.55 (1H, d, J=9.4 Hz, H-22), 2.31 (1H,m, H₁-21), 2.29 (1H, m, H-37), 2.03 (1H, br d, J=13.3 Hz, H₁-20), 1.92(3H, s, H-39), 1.76 (3H, s, H-34), 1.70 (3H, s, H-25), 1.69 (3H, s,H-35), 1.62 (3H, s, H-29), 1.60 (1H, m, H₂-20), 1.49 (3H, s, H-19), 1.39(1H, m, H₂-21), 1.31 (2H, m, H-36), 1.29 (3H, s, H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 204.09 (C-12), 178.99 (C-8), 168.63 (C-30),163.38 (C-18), 162.82 (C-6), 155.99 (C-16), 146.45 (C-38), 136.18(C-27), 134.28 (C-10, C-9), 131.50 (C-33), 128.27 (C-28), 122.31 (C-32),109.58 (C-40), 106.42 (C-17), 100.23 (C-5), 100.12 (C-7), 90.17 (C-14),84.66 (C-23), 83.64 (C-13), 79.48 (C-2), 71.37 (C-3), 48.90 (C-22),48.27 (C-37), 47.01 (C-11), 38.29 (C-20), 36.69 (C-4), 29.93 (C-25),29.47 (C-26), 28.85 (C-24), 25.73 (C-35), 25.14 (C-21), 23.66 (C-19),22.95 (C-39), 21.98 (C-36), 21.92 (C-31), 20.93 (C-29), 18.15 (C-34).

The EIMS data of Product Gh-1050 show a molecular ion peak [M]⁺ at m/z644 and a base peak at m/z 490 (100), and the HREIMS data of ProductGh-1050 show [M]⁺ at m/z 644.2983, indicating that the fragmentationpattern of Product Gh-1050 is similar to that of Product Gh-2603-2(namely, gambogellic acid), except for having sixteen more mass unitsthan gambogellic acid. Besides, the ¹H-NMR and ¹³C-NMR data of ProductGh-1050 are generally similar to those of Product Gh-2603-2.

The ¹H-NMR, ¹³C-NMR and ¹H-¹H COSY data of Product Gh-1050 show that:the structure of Product Gh-1050 includes a monoterpene moiety havingexo-methylene protons (δ 4.63 (H₁-40), δ 4.29 (H₂-40), and δ 109.58(C-40)) in an isopropenyl group, two adjacent methylene groups (δ 2.03(1H, br d, J=13.3 Hz, H₁-20), δ 1.60 (1H, m, H₂-20), δ 38.29 (C-20), δ1.31 (2H, m, H-36), and δ 21.98 (C-36)), a secondary hydroxy group (δ3.86 (1H, d, J=3.4 Hz, H-3) and δ 71.37 (C-3)), and a methine group (δ3.65 (1H, t, J=2.6 Hz, H-4) and δ 36.69 (C-4)). Besides, thehydroxymethine proton (δ 3.86 (1H, d, J=3.4 Hz, H-3)) and the methineproton (δ 3.65 (1H, t, J=2.6 Hz, H-4)) are adjacent and are coupled toeach other.

The HMBC data of Product Gh-1050 show that: the hydroxymethine proton (δ3.86 (1H, d, J=3.1 Hz, H-3)) is correlated with not only two quaternarycarbons (δ 79.48 (C-2) and δ 100.23 (C-5)) but also two methine groups(δ 36.69 (C-4) and δ 48.27 (C-37)); and δ 3.65 (H-4) is correlated notonly with δ 21.98 (C-38), δ 48.27 (C-37), δ 79.48 (C-2), δ 100.23 (C-5),δ 162.82 (C-6) and δ 100.12 (C-7), but also with δ 71.37 (C-3). Inaddition, the methylene protons (δ 2.03 (H₁-20) and δ 1.60 (H₂-20)) arecorrelated with δ 79.48 (C-2), δ 48.27 (C-37), δ 23.66 (C-19) and δ71.37 (C-3); and δ 1.49 (H-19) is correlated with δ 79.48 (C-2), δ 71.37(C-3), δ 21.98 (C-36) and an aromatic carbon (δ 163.38 (C-18)).Accordingly, it can be known that the hydroxyl group is connected toC-3; C-2 is connected to C-18 of an aromatic ring via an ether bond; andC-4 is connected to C-5 of the aromatic ring.

The NOESY data of Product Gh-1050 show that: δ 7.47 (H-10) is correlatedwith δ 3.49 (H-11); δ 3.49 (H-11) is correlated with δ 2.31 (H₁-21); δ1.39 (H₂-21) is correlated with δ 2.55 (H-22); both δ 2.31 (H₁-21) and δ2.55 (H-22) are correlated with δ 1.29 (H-24); and δ 5.57 (H-27) iscorrelated with δ 1.62 (H-29), evidencing that: the stereostructure ofProduct Gh-1050 in this part is identical to that of gambogic acid orgambogellic acid, i.e., in a 11S,13R,14S,22S configuration, whichincludes H-27 and carboxyl group (C-30) in a trans relationship, and adouble bond Δ^(27,28) in a Z configuration. The NOESY data of ProductGh-1050 also reveal that in addition to being correlated with δ 3.65(H-4) and δ 1.31 (H-36), δ 2.29 (H-37) is correlated with δ 3.86 (H-3)and δ 1.60 (H₂-20), evidencing that the monoterpene ring has a chairconformation with 1,3-diaxial interaction, in which H-37 and H-3 are inan axial orientation, and both the methyl group (C-19) and theisopropenyl group are in an equatorial orientation. Since thehydroxymethine proton (H-3) is in an axial orientation, the C-3 hydroxylgroup is in an equatorial orientation (an α-orientation), and C-3 is ina S configuration. In addition, since δ 1.62 (H-29) is not correlatedwith δ 4.63 (H₁-40) and δ 4.29 (H₂-40), C-2 is in an R configuration. Inview of the aforesaid, it can be known that the monoterpene moiety is ina 2R,3S,4R,37S configuration.

Based on the above information, Product Gh-1050 is identified to be anew compound having the following chemical structure:

Product Gh-1050 is identified by the name “3α-hydroxygambogellic acid”{IUPAC nomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,9R,10S,13R,16aS,17S)-3a,4,5,7,10,11,12,13-octahydro-8,17-dihydroxy-3,3,13-trimethyl-15-(3-methyl-2-butenyl)-10-(1-methylethenyl)-7,18-dioxo-1,5:9,13-dimethano-1H,3H,9H-furo[3.4-g]oxocino[3.2-b]xanthen-1-yl]-(2Z)-]}.

25. Product Gh-3291:

Product Gh-3291, which was purified from eluate 7 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 103˜106° C.

EIMS m/z (relative intensity): 644 [M]⁺ (10), 626 (10), 616 (12), 545(67), 517 (19), 471 (15), 459 (6), 419 (10), 389 (21), 349 (11), 347(20), 309 (9), 295 (17), 271 (13), 253 (20), 245 (26), 227 (21), 215(56), 189 (23), 171 (11), 147 (16), 129 (28), 105 (35), 99 (50), 84(43), 69 (84), 55 (100); HREIMS [M]⁺ m/z: 644.2991; calculated forC₃₈H₄₄O₉, 644.2985.

¹H-NMR (600 MHz, CDCl₃): δ 12.76 (1H, s, OH-6), 7.53 (1H, d, J=7.0 Hz,H-10), 6.60 (1H, d, J=10.2 Hz, H-4), 6.06 (1H, t, J=7.6 Hz, H-27), 5.36(1H, d, J=10.3 Hz, H-3), 5.00 (1H, brt, J=6.3 Hz, H-32), 4.89 (1H, s,H₁-40), 4.81 (1H, d, J=1.0 Hz, H₂-40), 4.00 (1H, t, J=6.2 Hz, H-37),3.46 (1H, dd, J=6.4, 5.1 Hz, H-11), 3.28 (1H, dd, J=14.9, 8.1 Hz,H₁-31), 3.13 (1H, br dd, J=14.6, 4.7 Hz, H₂-31), 2.93 (1H, dd, J=16.3,7.9 Hz, H₁-26), 2.87 (1H, dd, J=16.0, 7.6 Hz, H₂-26), 2.51 (1H, d, J=9.3Hz, H-22), 2.30 (1H, dd, J=13.5, 4.7 Hz, H₁-21), 1.80 (1H, m, H₁-20),1.73 (3H, s, H-29), 1.71 (3H, s, H-25), 1.68 (3H, s, H-34), 1.67 (1H, m,H₁-36), 1.66 (3H, m, H-39), 1.62 (3H, s, H-35), 1.61 (1H, m, H₂-36),1.54 (1H, m, H₂-20), 1.39 (3H, s, H-19), 1.38 (1H, m, H₂-21), 1.27 (3H,s, H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 203.19 (C-12), 178.90 (C-8), 169.81 (C-30),161.29 (C-18), 157.61 (C-16), 157.42 (C-6), 147.09 (C-38), 137.26(C-27), 135.40 (C-10), 133.31 (C-9), 131.65 (C=33), 127.90 (C-28),124.41 (C-3), 122.27 (C-32), 116.10 (C-4), 111.34 (C-40), 107.64 (C-17),102.63 (C-5), 100.50 (C-7), 90.91 (C-14), 84.03 (C-23), 83.73 (C-13),81.07 (C-2), 75.80 (C-37), 49.00 (C-22), 46.83 (C-11), 37.71 (C-20),29.82 (C-25), 29.29 (C-36), 29.05 (C-26), 28.83 (C-24), 27.73 (C-19),25.63 (C-35), 25.15 (C-21), 21.58 (C-31), 20.76 (C-29), 18.11 (C-34),17.45 (C-39).

The EIMS data of Product Gh-3291 show a molecular ion peak [M]⁺ at m/z644, and the HREIMS data of Product Gh-3291 show [M]⁺ at m/z 644.2991,indicating that Product Gh-3291 has a molecular formula of C₃₈H₄₄O₉,which has 16 more mass units than the molecular formula of gambogicacid.

The ¹H-NMR, ¹³C-NMR and HMQC data of Product Gh-3291 show that ProductGh-3291 has a C-2 side chain different from that of gambogic acid,namely, a 3-hydroxy-4-methyl-4-pentenyl group in lieu of the4-methyl-3-pentenyl group in gambogic acid. The ¹H-NMR and ¹³C-NMR dataof Product Gh-3291 also reveal that Product Gh-3291 has a secondaryhydroxy group (δ 4.00 (1H, t, J=6.2 Hz) and δ 75.80) and anexo-methylene group (δ 4.89 (1H, s), δ 4.81 (1H, d), and δ 111.34).

The HMBC data of Product Gh-3291 show that: in addition to beingcorrelated with δ 111.34 (C-40) and δ 147.09 (C-38), the hydroxymethineproton (δ 4.00) is correlated with δ 17.45 (C-39), δ 29.29 (C-36) and δ37.71 (C-20); in addition to being correlated with δ 37.71 (C-20), themethyl proton (δ 1.39 (H-19)) is correlated with δ 81.07 (C-2) and δ124.41 (C-3); and δ 5.36 (H-3) is correlated with δ 81.07 (C-2), δ 27.73(C-19) and δ 37.71 (C-20).

The ¹H-¹H COSY data of Product Gh-3291 show that: δ 4.00 (1H, t, J=6.2Hz, H-37) is coupled to the methylene protons (δ 1.67 (H₁-36) and δ 1.61(H₂-36)), evidencing that the hydroxy group is attached to C-37.

The NOESY data of Product Gh-3291 show that: δ 7.53 (H-10) is correlatedwith δ 3.46 (H-11); δ 3.46 (H-11) is correlated with δ 2.30 (H₁-21); δ1.38 (H₂-21) is correlated with δ 2.51 (H-22) and δ 1.27 (H-24); and δ6.06 (H-27) is correlated with 581.73 (H-29), evidencing that thestereostructure of Product Gh-3291 in this part is identical to that ofgambogic acid, i.e., in a 11S,13R,14S,22S configuration, which includesH-27 and carboxyl group (C-30) in a trans relationship, and a doublebond Δ^(27,28) in a Z configuration.

The NOESY data of Product Gh-3291 also reveal that: the hydroxymethineproton (δ 4.00 (H-37)) is correlated not only with δ 4.89 (H₁-40) and δ1.66 (H-39), but also with the protons of two methylene groups (δ 1.80(H₁-20), δ 1.54 (H₂-20), δ 1.67 (H₁-36) and δ 1.61 (H₂-36)); and δ 5.36(H-3) is correlated with δ 1.39 (H-19), δ 1.80 (H₁-20), δ 1.54 (H₂-20),δ 1.67 (H₁-36), and δ 1.61 (H₂-36). Besides, since δ 1.39 (H-19) iscorrelated with δ 1.73 (H-29), C-2 is in an R configuration.

Based on the above information, Product Gh-3291 is identified to be anew compound having the following chemical structure:

Product Gh-3291 is identified by the name “formoxanthone D” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11R,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(3-methyl-2-butenyl)-11-(3-hydroxy-4-methyl-4-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

26. Product Gh-3352:

Product Gh-3352, which was purified from eluate 12 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 194˜197° C.

FABMS m/z (relative intensity): 645 [M+H]⁺ (11), 634 (10), 591 (11), 575(13), 574 (22), 573 (45), 559 (12), 545(16), 544 (11), 487 (15), 417(16), 405 (11), 391 (13), 375 (10), 371 (10), 349 (11), 338 (12), 321(12), 307 (27), 299 (17), 295 (17), 289 (23), 259 (19), 255 (21), 219(24), 215 (20), 213(27), 189 (18), 176 (21), 165 (24), 154(100),136(92), 121 (30), 107 (56), 91 (55), 89 (50), 77 (59), 69 (69), 57(69), 55 (66); HRFABMS [M+H]⁺ m/z 645.3066; calculated for C₃₈H₄₅O₉,645.3064.

¹H-NMR (600 MHz, CDCl₃): δ 12.92 (1H, s, OH-6), 7.52 (1H, d, J=6.9 Hz,H-10), 6.66 (1H, d, J=10.1 Hz, H-4), 5.58 (1H, br t, J=6.5 Hz, H-27),5.45 (1H, d, J=10.2 Hz, H-3), 5.03 (1H, t, J=7.0 Hz, H-37), 3.79 (1H,dd, J=9.2, 3.1 Hz, H-32), 3.50 (1H, dd, J=6.8, 4.7 Hz, H-11), 3.30 (1H,dd, J=14.8, 10.1 Hz, H₁-26), 2.90 (1H, ddd, J=15.3, 5.6, 1.3 Hz, H₂-26),2.85 (1H, dd, J=13.9, 9.7 Hz, H₁-31), 2.71 (1H, dd, J=13.8, 3.4 Hz,H₂-31), 2.49 (1H, d, J=9.3 Hz, H-22), 2.33 (1H, dd, J=13.5, 4.7 Hz,H₁-21), 2.05 (2H, m, H-36), 1.74 (3H, s, H-25), 1.73 (1H, m, H₁-20),1.69 (3H, s, H-29), 1.66 (1H, m, H₂-20), 1.63 (3H, s, H-39), 1.53 (3H,s, H-40), 1.45 (3H, s, H-19), 1.33 (1H, m, H₂-21), 1.29 (3H, s, H-34),1.27 (3H, s, H-35), 1.25 (3H, s, H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 202.89 (C-12), 179.05 (C-8), 168.89 (C-30),161.34 (C-18), 158.49 (C-6), 158.28 (C-16), 136.09 (C-27), 135.01(C-10), 133.19 (C-9), 132.17 (C-38), 129.31 (C-28), 124.75 (C-3), 123.46(C-37), 115.93 (C-4), 104.83 (C-17), 102.92 (C-5), 100.67 (C-7), 90.77(C-14), 84.28 (C-13), 83.86 (C-23), 81.81 (C-2), 77.20 (C-32), 73.24(C-33), 49.12 (C-22), 47.04 (C-11), 41.72 (C-20), 30.56 (C-25), 29.65(C-26), 28.89 (C-24), 27.18 (C-19), 25.89 (C-31), 25.69 (C-39), 25.63(C-35), 25.36 (C-21), 23.75 (C-34), 22.66 (C-36), 20.73 (C-29), 17.59(C-40).

The HRFABMS data of Product Gh-3352 show a pseudomolecular ion peak[M+H]⁺ at m/z 645.3066, suggesting that Product Gh-3352 has a molecularformula of C₃₈H₄₄O₉, which has sixteen more mass units than themolecular formula of gambogic acid.

The ¹H-NMR, ¹³C-NMR and HMQC data of Product Gh-3352 show that ProductGh-3352 has a C-17 side chain different from that of gambogic acid,namely, a 2,3-epoxy-3-methylbutyl group in lieu of the3-methyl-2-butenyl group in gambogic acid. As compared to gambogic acid,the unsaturation number of Product Gh-3352 remains unchanged.

The ¹H-¹H COSY, HMQC and HMBC data of Product Gh-3352 show that: anoxymethine proton (δ 3.79 (1H, dd, J=9.2, 3.1 Hz)) is coupled to amethylene proton (δ 2.85 (1H, dd, J=13.9, 9.7 Hz, H₁-31)); and δ 2.85 iscoupled to another methylene proton (δ 2.71 (1H, dd, J=13.8, 3.4 Hz,H₂-31)). Besides, Product Gh-3352 has two tertiary methyl groups (δ 1.29(3H, s, H-34) and δ 1.27 (3H, s, H-35)) attached to an oxygen-bearingquaternary carbon. It can be known from the signals of theoxygen-bearing quaternary carbon (δ 73.24 (C-33)), the oxymethine carbon(δ 77.20 (C-32) and the methylene carbon (δ 25.89 (CH₂), C-31) that anepoxy group is located at C-32 and C-33.

The HMBC data of Product Gh-3352 show that δ 2.85 (H₁-31) is correlatedwith δ 77.20 (C-32) and δ 104.83 (C-17), evidencing that the sideattached to C-17 is a 2,3-epoxy-3-methylbutyl group.

The NOESY data of Product Gh-3352 show that: δ 7.52 (H-10) is correlatedwith δ 3.50 (H-11); δ 3.50 (H-11) is correlated with δ 2.33 (H₁-21) andδ 1.33 (H₂-21); δ 1.33 (H₂-21) and δ 2.49 (H-22) are correlated; δ 2.49(H-22) is correlated with δ 1.74 (H-25); and δ 5.58 (H-27) is correlatedwith δ 1.69 (H-29), evidencing that the stereostructure of ProductGh-3352 in this part is identical to that of gambogic acid, i.e., in a11S,13R,14S,22S configuration, which includes H-27 and carboxyl group(C-30) in a trans relationship, and a double bond Δ^(27,28) in a Zconfiguration. In addition, since δ 1.69 (H-29) is correlated with δ1.45 (H-19), C-2 is in an R configuration.

Based on the above information, Product Gh-3352 is identified to be anew compound having the following chemical structure:

Product Gh-3352 is identified by the name “formoxanthone E” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11R,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(2,3-epoxy-3-methylbutyl)-11-(4-methyl-3-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

27. Product Gh-3351:

Product Gh-3351, which was purified from eluate 10 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 168˜171° C.

EIMS m/z (relative intensity): 621 (6), 603 (17), 589 (100), 577(23),561 (19), 503 (47), 467 (6), 423 (8), 381 (5), 339 (4), 315 (15), 231(7), 213 (9), 135 (4), 69 (15); HRFABMS [M+H]⁺ m/z: 645.3070; calculatedfor C₃₈H₄₅O₉, 645.3064.

¹H-NMR (600 MHz, CDCl₃): δ 12.91 (1H, s, OH-6), 7.51 (1H, d, J=6.9 Hz,H-10), 6.49 (1H, d, J=10.1 Hz, H-4), 5.56 (1H, br t, J=7.3 Hz, H-27),5.43 (1H, d, J=10.2 Hz, H-3), 5.03 (1H, t, J=7.0 Hz, H-37), 3.76 (1H, brd, J=7.0 Hz, H-32), 3.50 (1H, dd, J=6.6, 4.9 Hz, H-11), 3.29 (1H, dd,J=15.2, 10.1 Hz, H₁-26), 2.89 (1H, ddd, J=15.3, 5.5, 1.5 Hz, H₂-26),2.82 (1H, dd, J=13.9, 9.9 Hz, H₁-31), 2.69 (1H, br d, J=12.7 Hz, H₂-31),2.48 (1H, d, J=9.3 Hz, H-22), 2.32 (1H, dd, J=13.5, 4.0 Hz, H₁-21), 2.03(2H, m, H-36), 1.73 (3H, s, H-25), 1.68 (3H, s, H-29), 1.67 (2H, m,H-20), 1.62 (3H, s, H-39), 1.53 (3H, s, H-40), 1.43 (3H, s, H-19), 1.30(1H, in, H₂-21), 1.28 (3H, s, H-34), 1.26 (3H, s, H-35), 1.24 (3H, s,H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 202.99 (C-12), 179.03 (C-8), 169.60 (C-30),161.37 (C-18), 158.46 (C-6), 158.25 (C-16), 135.76 (C-27), 134.92(C-10), 133.21 (C-9), 132.14 (C-38), 129.58 (C-28), 124.72 (C-3), 123.47(C-37), 115.90 (C-4), 104.81 (C-17), 102.88 (C-5), 100.63 (C-7), 90.74(C-14), 84.26 (C-13), 83.79 (C-23), 81.75 (C-2), 77.15 (C-32), 73.25(C-33), 49.11 (C-22), 47.04 (C-11), 41.71 (C-20), 30.56 (C-25), 29.69(C-26), 28.88 (C-24), 27.13 (C-19), 25.92 (C-31), 25.89 (C-39), 25.63(C-35), 25.36 (C-21), 23.44 (C-34), 22.65 (C-36), 20.74 (C-29), 17.59(C-40).

The EIMS data of Product Gh-3351 show a base peak at m/z 589 (100), andthe HRFABMS data of Product Gh-3351 show a pseudomolecular ion peak[M+H]⁺ at m/z 645.3070, indicating that Product Gh-3351 has a molecularformula of C₃₈H₄₄O₉, which has sixteen more mass units than themolecular formula of gambogic acid.

The ¹H-NMR, ¹³C-NMR and HMQC data of Product Gh-3351 show that ProductGh-3351 has a C-17 side chain different from that of gambogic acid,namely a 2,3-epoxy-3-methylbutyl group in lieu of the 3-methyl-2-butenylgroup in gambogic acid. As compared to gambogic acid, the unsaturationnumber of Product Gh-3351 remains unchanged.

The ¹H-¹H COSY, HMQC and HMBC data of Product Gh-3351 show that: anoxymethine proton (δ 3.76 (1H, br d, J=7.0 Hz)) is coupled to amethylene proton (δ 2.82 (1H, dd, J=13.9, 9.9 Hz, H₁-31)); and δ 2.82 iscoupled to another methylene proton (δ 2.69 (1H, br d, J=12.7 Hz,H₂-31)). Besides, Product Gh-3351 has two oxygen-bearing tertiary methylprotons (δ 1.28 (3H, s, H-34) and δ 1.26 (3H, s, H-35)]. Based on thesignals of the oxygen-bearing quaternary carbon (δ 73.25 (C-33)), theoxymethine group (δ 77.15 (C-32)) and the methylene group (δ 25.92(C-31)), it can be known that an epoxy group is located at C-32 andC-33.

The HMBC data of Product Gh-3351 show that: δ 2.82 (H₁-31) is correlatedwith δ 77.15 (C-32), δ 102.88 (C-5), δ 104.81 (C-17), δ 158.25 (C-16)and δ 161.37 (C-18), evidencing that the side chain attached to C-17 isa 2,3-epoxy-3-methylbutyl group.

The NOESY data of Product Gh-3351 show that: δ 7.51 (H-10) is correlatedwith δ 3.50 (H-11); δ 3.50 (H-11) is correlated with δ 2.32 (H₁-21) andδ 1.30 (H₂-21); δ 1.30 (H₂-21) is correlated with δ 2.48 (H-22); δ 2.48(H-22) is correlated with δ 1.73 (H-25); and δ 5.56 (H-27) is correlatedwith δ 1.68 (H-29), evidencing that the stereo structure of ProductGh-3351 in this part is identical to that of gambogic acid, i.e., in a11S,13R,14S,22S configuration, which includes H-27 and carboxyl group(C-30) in a trans relationship, and a double bond Δ^(27,28) in a Zconfiguration. In addition, since δ 1.68 (H-29) is correlated with δ2.03 (H-36), C-2 is in an S configuration.

Based on the above information, product Gh-3351 is identified to be anew compound having the following chemical structure:

Product Gh-3351 is identified by the name “epiformoxanthone E” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11S,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(2,3-epoxy-3-methylbutyl)-11-(4-methyl-3-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

28. Product Gh-1052:

Product Gh-1052, which was purified from eluate 9 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 83˜85° C.

EIMS m/z (relative intensity): 662 [M]⁺ (6), 634 (8), 579 (100), 551(16), 545 (9), 507 (14), 489 (6), 417 (12), 389 (4), 349 (4), 295 (5),245 (8), 214.9 (14), 189 (8), 147 (4), 99 (7), 69 (29); HREIMS [M]⁺ m/z:662.3096; calculated for C₃₈H₄₆O₁₀, 662.3091.

¹H-NMR (600 MHz, CDCl₃): δ 12.92 (1H, s, OH-6), 7.52 (1H, d, J=7.0 Hz,H-10), 6.65 (1H, d, J=10.2 Hz, H-4), 5.63 (1H, t, J=7.4 Hz, H-27), 5.46(1H, d, J=10.3 Hz, H-3), 5.07 (1H, t, J=7.1 Hz, H-37), 3.74 (1H, dd,J=10.0, 3.4 Hz, H-32), 3.49 (1H, dd, J=6.7, 4.7 Hz, H-11), 3.25 (1H, dd,J=15.2, 9.9 Hz, H₁-26), 2.89 (1H, ddd, J=15.3, 5.9, 1.7 Hz, H₂-26), 2.84(1H, dd, J=13.9, 10.1 Hz, H₁-31), 2.72 (1H, dd, J=13.9, 3.4 Hz, H₂-31),2.49 (1H, d, J=9.3 Hz, H-22), 2.32 (1H, dd, J=13.5, 4.7 Hz, H₁-21), 2.07(2H, m, H-36), 1.79 (1H, m, H₁-20), 1.73 (3H, s, H-25), 1.69 (3H, s,H-29), 1.67 (1H, m, H₂-20), 1.65 (3H, s, H-39), 1.57 (3H, s, H-40), 1.37(3H, s, H-19), 1.33 (1H, dd, J=13.5, 9.6 Hz, H₂-21), 1.27 (3H, s, H-35),1.26 (3H, s, H-24), 1.24 (3H, s, H-34).

¹³C-NMR (150 MHz, CDCl₃): δ 202.93 (C-12), 179.10 (C-8), 169.18 (C-30),161.47 (C-18), 158.49 (C-6), 158.22 (C-16), 136.45 (C-27), 135.16(C-10), 133.12 (C-9), 132.66 (C-38), 129.08 (C-28), 124.56 (C-3), 123.36(C-37), 116.12 (C-4), 104.92 (C-17), 102.95 (C-5), 100.63 (C-7), 90.82(C-14), 84.18 (C-13), 83.82 (C-23), 81.76 (C-2), 77.11 (C-32), 73.09(C-33), 49.08 (C-22), 47.03 (C-11), 41.74 (C-20), 30.62 (C-25), 29.54(C-26), 28.83 (C-24), 27.21 (C-19), 25.87 (C-35), 25.68 (C-31), 25.65(C-39), 25.33 (C-21), 23.65 (C-34), 22.91 (C-36), 20.74 (C-29), 17.71(C-40).

The EIMS data of Product Gh-1052 show a molecular ion peak [M]⁺ at m/z662 and a base peak at m/z 579 (100), and the HREIMS data of ProductGh-1052 show [M]4 at m/z 662.3096, indicating that Product Gh-1052 has amolecular formula of C₃₈H₄₆O₁₀, which has thirty-four more mass unitsthan the molecular formula of gambogic acid.

The ¹H-NMR, ¹³C-NMR and HMQC data of Product Gh-1052 show that ProductGh-1052 has a C-17 side chain different from that of gambogic acid,namely, a 2,3-dihydroxy-3-methylbutyl group in lieu of the3-methyl-2-butenyl group in gambogic acid.

The ¹H-NMR and ¹³C-NMR data of Product Gh-1052 show that Product Gh-1052has a secondary hydroxy group (δ 3.74 (1H, dd, J=10.0, 3.4 Hz) and δ77.11] and an oxygen-bearing quaternary carbon (δ 73.09). In addition,the ¹H-¹H COSY data of Product Gh-1052 show that a hydroxymethine proton(δ 3.74 (1H, dd, J=10.0, 3.4 Hz)) is coupled to two methylene protons (δ2.84 (1H, dd, J=13.9, 10.1 Hz, H₁-31) and δ 2.72 (1H, dd, J=13.9, 3.4Hz, H₂-31)).

The HMQC data of Product Gh-1052 show that: C-31 (δ 25.68) is amethylene group; the secondary hydroxy group is connected to C-32 (δ77.11); a tertiary hydroxy group is connected to C-33 (δ 73.09); andC-35 (δ 25.87) and C-34 (δ 23.65) are tertiary methyl groups.

The HMBC data of Product Gh-1052 show that: δ 3.74 (H-32) is correlatedwith δ 73.09 (C-33) and δ 23.65 (C-34); both δ 2.84 (H₁-31) and δ 2.72(H₂-31) are correlated with δ 104.92 (C-17), δ 161.47 (C-18), δ 158.22(C-16) and δ 77.11 (C-32); H₁-31 is also correlated with δ 73.09 (C-33);δ 1.24 (H-34) and δ 1.27 (H-35) are both correlated with δ 77.11 (C-32)and δ 73.09 (C-33); H-34 is also correlated with δ 25.87 (C-35); andH-35 is correlated with δ 23.65 (C-34), evidencing that the side chainattached to C-17 is a 2,3-dihydroxy-3-methylbutyl group.

The NOESY data of Product Gh-1052 show that: δ 7.52 (H-10) is correlatedwith δ 3.49 (H-11); δ 3.49 (H-11) is correlated with δ 2.32 (H₁-21) andδ 1.33 (H₂-21); δ 1.33 (H₂-21) is correlated with δ 2.49 (H-22); δ 2.49(H-22) is correlated with δ 1.73 (H-25); and δ 5.63 (H-27) is correlatedwith δ 1.69 (H-29), evidencing that the stereostructure of ProductGh-1052 in this part is identical to that of gambogic acid, i.e., in a11S,13R,14S,22S configuration, which includes H-27 and carboxyl group(C-30) in a trans relationship, and a double bond Δ^(27,28) in a Zconfiguration. In addition, since δ 1.37 (H-19) is correlated with δ1.69 (H-29), C-2 is in an R configuration.

Based on the above information, Product Gh-1052 is identified to be anew compound having the following chemical structure:

Product Gh-1052 is identified by the name “formoxanthone F” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11R,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(2,3-dihydroxy-3-methylbutyl)-11-(4-methyl-3-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

29. Product Gh-1036:

Product Gh-1036, which was purified from eluate 6 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 76˜78° C.

EIMS m/z (relative intensity): 662 [M]⁺ (13), 634 (7), 579 (100), 551(13), 545 (10), 507 (12), 489 (5), 417 (9), 375 (3), 349 (3), 295 (4),245 (8), 215 (8), 213 (7), 147 (4), 105 (5), 69 (19); HREIMS [M]⁺ m/z662.3098; calculated for C₃₈H₄₆O₁₀, 662.3091.

¹H-NMR (600 MHz, CDCl₃): δ 12.91 (1H, s, OH-6), 7.52 (1H, d, J=6.9 Hz,H-10), 6.66 (1H, d, J=10.0 Hz, H-4), 5.53 (1H, br t, J=5.2 Hz, H-27),5.47 (1H, d, J=10.1 Hz, H-3), 5.08 (1H, t, J=6.9 Hz, H-37), 3.74 (1H,dd, J=11.2, 2.6 Hz, H-32), 3.49 (1H, t, J=5.6 Hz, H-11), 3.27 (1H, dd,J=15.0, 10.4 Hz, H₁-26), 2.87 (1H, dd, J=13.6, 11.7 Hz, H₁-31), 2.82(1H, br dd, J=15.3, 3.8 Hz, H₂-26), 2.72 (1H, dd, J=13.7, 2.8 Hz,H₂-31), 2.48 (1H, d, J=9.2 Hz, H-22), 2.32 (1H, dd, J=13.5, 4.5 Hz,H₁-21), 2.10 (2H, m, H-36), 1.78 (1H, m, H₁-20), 1.702 (3H, s, H-25),1.697 (1H, m, H₂-20), 1.653 (3H, s, H-29), 1.648 (3H, s, H-39), 1.58(3H, s, H-40), 1.35 (3H, s, H-19), 1.33 (1H, m, H₂-21), 1.28 (3H, s,H-35), 1.25 (3H, s, H-24), 1.24 (3H, s, H-34).

¹³C-NMR (150 MHz, CDCl₃): δ 202.96 (C-12), 179.04 (C-8), 168.66 (C-30),161.45 (C-18), 158.35 (C-6), 158.28 (C-16), 135.28 (C-27), 134.98(C-10), 133.19 (C-9), 132.39 (C-38), 129.46 (C-28), 124.77 (C-3), 123.52(C-37), 116.07 (C-4), 104.54 (C-17), 103.19 (C-5), 100.64 (C-7), 90.87(C-14), 84.28 (C-13), 83.60 (C-23), 81.71 (C-2), 76.57 (C-32), 73.06(C-33), 49.07 (C-22), 46.99 (C-11), 41.68 (C-20), 30.76 (C-25), 29.96(C-26), 28.86 (C-24), 26.79 (C-19), 26.29 (C-35), 25.63 (C-39), 25.42(C-21), 24.62 (C-31), 23.56 (C-34), 22.82 (C-36), 20.52 (C-29), 17.62(C-40).

The EIMS data of Product Gh-1036 show a molecular ion peak [M]⁺ at m/z662 and a base peak at m/z 579 (100), and the HREIMS data of ProductGh-1036 show [M]⁺ at m/z 662.3098, indicating that Product Gh-1036 has amolecular formula of C₃₈H₄₆O₁₀, which has thirty-four more mass unitsthan the molecular formula of gambogic acid.

The ¹H-NMR, ¹³C-NMR and HMQC data of Product Gh-1036 show that ProductGh-1036 has a C-17 side chain different from that of gambogic acid,namely, a 2,3-dihydroxy-3-methylbutyl group in lieu of the3-methyl-2-butenyl group in gambogic acid.

The ¹H-NMR and ¹³C-NMR data of Product Gh-1036 show that Product Gh-1036has a secondary hydroxy group (δ 3.74 (1H, dd, J=11.2, 2.6 Hz) and δ76.57) and an oxygen-bearing quaternary carbon (δ 73.06).

In addition, the ¹H-¹H COSY data of Product Gh-1036 show that ahydroxymethine proton (δ 3.74 (1H, dd, J=11.2, 2.6 Hz)) is coupled totwo methylene protons (S 2.87 (1 hi, dd, J=13.6, 11.7 Hz, H₁-31) and δ2.72 (1H, dd, J=13.7, 2.8 Hz, H₂-31)).

The HMQC data of product Gh-1036 show that: C-31 (δ 24.62) is amethylene group; a secondary hydroxy group is connected to C-32 (δ76.57); a tertiary hydroxy group is connected to C-33 (δ 73.06); andC-34 (δ 23.56) and C-35 (δ 26.29) are tertiary methyl groups.

The HMBC data of Product Gh-1036 show that: in addition to beingcorrelated with δ 24.62 (C-31), δ 73.06 (C-33) and δ 23.56 (C-34), δ3.74 (H-32) is correlated with δ 104.54 (C-17); and both δ 2.87 (H₁-31)and δ 2.72 (H₂-31) are correlated with δ 76.57 (C-32), δ 73.06 (C-33), δ104.54 (C-17), δ 161.45 (C-18) and δ 158.28 (C-16), evidencing that theside chain attached to C-17 is a 2,3-dihydroxy-3-methylbutyl group.

The NOESY data of Product Gh-1036 show that: δ 7.52 (H-10) is correlatedwith δ 3.49 (H-11); δ 3.49 (H-11) is correlated with δ 2.32 (H₁-21) andδ 1.33 (H₂-21); δ 1.33 (H₂-21) is correlated with δ 2.48 (H-22); δ 2.48(H-22) is correlated with δ 1.702 (H-25); and δ 5.53 (H-27) iscorrelated with δ 1.653 (H-29), evidencing that the stereostructure ofProduct Gh-1036 in this part is identical to that of gambogic acid,i.e., in a 11S,13R,14S,22S configuration, which includes H-27 andcarboxyl group (C-30) in a trans relationship, and a double bondΔ^(27,28) in a Z configuration. In addition, since δ 1.653 (H-29) iscorrelated with δ 1.78 (H₁-20) and δ 2.10 (H-36), C-2 is in an Sconfiguration.

Based on the above information, Product Gh-1036 is identified to be anew compound having the following chemical structure:

Product Gh-1036 is identified by the name “epiformoxanthone F” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11S,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(2,3-dihydroxy-3-methylbutyl)-11-(4-methyl-3-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

30. Product Gh-3353:

Product Gh-3353, which was purified from eluate 11 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 158˜162° C.

EIMS m/z (relative intensity): 590 (32), 589 (100), 577 (24), 561 (19),503 (44), 467 (6), 423 (8), 381 (5), 339 (4), 315 (8), 311 (3), 285 (2),247 (4), 231 (6), 205 (2), 135 (4), 81 (3), 69 (14); HRFABMS [M+H]⁺ m/z:661.3019; calculated for C₃₈H₄₅O₁₀, 661.3013.

¹H-NMR (600 MHz, CDCl₃): δ 13.13 (1H, s, OH-6), 7.56 (1H, d, J=7.1 Hz,H-10), 6.62 (1H, d, J=10.1 Hz, H-4), 5.44 (1H, d, J=10.3 Hz, H-3), 5.15(1H, d, J=8.9 Hz, H-31), 5.04 (1H, br t, J=3.5 Hz, H-37), 5.01 (1H, m,H-27), 4.70 (1H, d, J=8.9 Hz, H-32), 3.55 (1H, dd, J=6.6, 4.9 Hz, H-11),3.42 (1H, t, J=13.4 Hz, H₁-26), 2.73 (1H, br d, J=13.9 Hz, H₂-26), 2.52(1H, d, J=9.3 Hz, H-22), 2.36 (1H, dd, J=13.5, 7.7 Hz, H₁-21), 2.05 (2H,m, H-36), 1.82 (3H, s, H-25), 1.76 (1H, m, H₁-20), 1.64 (3H, s, H-35),1.63 (1H, m, H₂-20), 1.62 (3H, s, H-39), 1.54 (3H, s, H-40), 1.49 (3H,s, H-29), 1.44 (6H, s, H-19, H-34), 1.27 (1H, m, H₂-21), 1.26 (3H, s,H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 202.26 (C-12), 179.10 (C-8), 166.33 (C-30),161.08 (C-18), 159.90 (C-16), 158.73 (C-6), 134.79 (C-10), 132.77 (C-9),132.73 (C-28), 132.09 (C-38), 128.48 (C-27), 124.60 (C-3), 123.55(C-37), 115.48 (C-4), 105.98 (C-17), 101.92 (C-5), 99.90 (C-7), 91.89(C-14), 84.63 (C-13), 83.75 (C-33), 83.70 (C-23), 82.16 (C-2), 76.83(C-32), 67.26 (C-31), 49.47 (C-22), 46.71 (C-11), 41.97 (C-20), 31.14(C-26), 30.13 (C-25), 29.42 (C-24), 27.60 (C-19), 26.05 (C-21), 25.63(C-39), 23.89 (C-35), 22.43 (C-36), 19.98 (C-29), 18.80 (C-34), 17.66(C-40).

The EIMS data of Product Gh-3353 show a base peak at m/z 589 (100), andthe HRFABMS data of product Gh-3353 show a pseudomolecular ion peak[M+H]⁺ at m/z 661.3019, indicating that Product Gh-3353 has a molecularformula of C₃₈H₄₄O₁₀, which has thirty-two more mass units than themolecular formula of gambogic acid.

The ¹H-NMR, ¹³C-NMR and HMQC data of Product Gh-3353 show that ProductGh-3353 has a C-17 side chain different from that of gambogic acid,namely, a 1-hydroxy-2,3-epoxy-3-methylbutyl group in lieu of the3-methyl-2-butenyl group in gambogic acid.

The ¹H-NMR, ¹³C-NMR and ¹H-¹H COSY data of Product Gh-3353 show that ahydroxymethine proton (δ 5.15 (1H, d, J=8.9 Hz)) is coupled to anoxymethine proton (δ 4.70 (1H, d, J=8.9 Hz)) on an epoxy group. Sincethe two vicinal oxymethine protons on an epoxy ring have a couplingconstant (J) normally less than 5 Hz, the coupling constant of J=8.9 Hzshould not represent the coupling of the two vicinal oxymethine protonson the epoxy ring.

The HMBC data of Product Gh-3353 show that: δ 5.15 is correlated with δ161.08 (C-18), δ 159.90 (C-16), δ 105.98 (C-17) and δ 76.83 (C-32); andδ 4.70 is correlated with δ 83.75 (C-33), δ 18.80 (C-34), δ 23.89 (C-35)and δ 67.26 (C-31), evidencing that a hydroxy group is connected toC-31; an epoxy group is located at C-32 and C-33; and the side chainattached to C-17 is a 1-hydroxy-2,3-epoxy-3-methylbutyl group.

The NOESY data of Product Gh-3353 show that: δ 7.56 (H-10) is correlatedwith δ 3.55 (H-11); δ 3.55 (H-11) is correlated with δ 2.36 (H₁-21) andδ 1.27 (H₂-21); both δ 2.36 (H₁-21) and δ 1.27 (H₂-21) are correlatedwith δ 2.52 (H-22); δ 2.52 (H-22) is correlated with δ 1.82 (H-25) and δ1.26 (H-24); δ 5.01 (H-27) is correlated with δ 1.49 (H-29); and δ 1.49(H-29) is not correlated with δ 2.05 (H-36) or S 1.76 (H₁-20),evidencing that the stereostructure of Product Gh-3353 in this part isidentical to that of gambogic acid, i.e., in a 11S,13R,14S,22Sconfiguration, which includes H-27 and carboxyl group (C-30) in a transrelationship, and a double bond Δ^(27,28) in a Z configuration. Inaddition, δ 5.04 (H-37) is correlated with δ 1.62 (H-39) and δ 1.54(H-40), and δ 2.05 (H-36) is correlated with δ 1.76 (H₁-20). Since δ1.49 (H-29) is not correlated with δ 2.05 (H-36) or δ 1.76 (H₁-20), C-2has an R configuration.

Based on the above information, Product Gh-3353 is identified to be anew compound having the following chemical structure:

Product Gh-3353 is identified by the name “formoxanthone G” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11R,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(1-hydroxy-2,3-epoxy-3-methylbutyl)-11-(4-methyl-3-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

31. Product Gh-3311:

Product Gh-3311, which was purified from eluate 4 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 144˜148° C.

EIMS m/z (relative intensity): 590 (7), 589 (18), 577 (5), 561 (9), 503(11), 467 (2), 423 (2), 347 (5), 315 (3), 285 (3), 247 (6), 233 (13),231 (22), 230 (16), 215 (8), 202 (4), 131 (6), 117 (100), 115 (19), 91(12), 69 (14); HRFABMS [M+H]⁺ m/z: 661.3010; calculated for C₃₈H₄₅O₁₀,661.3013.

¹H-NMR (600 MHz, CDCl₃): δ 13.12 (1H, s, OH-6), 7.56 (1H, d, J=7.1 Hz,H-10), 6.65 (1H, d, J=10.0 Hz, H-4), 5.52 (1H, d, J=10.2 Hz, H-3), 5.17(1H, d, J=9.0 Hz, H-31), 5.05 (1H, m, H-37), 5.03 (1H, m, H-27), 4.71(1H, d, J=9.0 Hz, H-32), 3.56 (1H, dd, J=6.8, 4.8 Hz, H-11), 3.42 (1H,t, J=13.5 Hz, H₁-26), 2.74 (1H, br d, J=13.7 Hz, H₂-26), 2.54 (1H, d,J=9.3 Hz, H-22), 2.37 (1H, dd, J=13.5, 4.8 Hz, H₁-21), 2.05 (2H, m,H-36), 1.86 (1H, m, H₁-20), 1.83 (3H, s, H-25), 1.69 (1H, m, H₂-20),1.66 (3H, s, H-39), 1.64 (3H, s, H-35), 1.56 (3H, s, H-40), 1.49 (3H, s,H-29), 1.45 (3H, s, H-34), 1.35 (3H, s, H-19), 1.28 (1H, m, H₂-21), 1.26(3H, s, H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 202.22 (C-12), 179.25 (C-8), 166.30 (C-30),160.99 (C-18), 159.85 (C-16), 158.80 (C-6), 134.86 (C-10), 132.81 (C-9),132.75 (C-28), 132.17 (C-38), 128.54 (C-27), 125.25 (C-3), 123.50(C-37), 116.08 (C-4), 106.57 (C-17), 102.68 (C-5), 100.01 (C-7), 91.92(C-14), 84.65 (C-13), 83.80 (C-33), 83.66 (C-23), 81.92 (C-2), 76.98(C-32), 67.16 (C-31), 49.47 (C-22), 46.75 (C-11), 41.64 (C-20), 31.16(C-26), 30.15 (C-25), 29.43 (C-24), 26.90 (C-19), 26.08 (C-21), 25.67(C-39), 23.83 (C-35), 23.21 (C-36), 19.89 (C-29), 18.91 (C-34), 17.72(C-40).

The EIMS data of Product Gh-3311 show a base peak at m/z 117 (100) butno molecular ion peak [M]⁺, and the HRFABMS data of Product Gh-3311 showa pseudomolecular ion peak [M+H]⁺ at m/z 661.3010, indicating thatProduct Gh-3311 has a molecular formula of C₃₈H₄₄O₁₀, which hasthirty-two more mass units compared to the molecular formula of gambogicacid. As compared to gambogic acid, the unsaturation number of ProductGh-3331 remains unchanged.

The ¹H-NMR and ¹³C-NMR data of Product Gh-3311 show that Product Gh-3311has a secondary hydroxy group (δ 5.17 (1H, d, J=9.0 Hz) and δ 67.16) andan epoxy group (δ 4.71 (1H, d, J=9.0 Hz) and δ 76.98). These ¹H-NMR and¹³C-NMR data of Product Gh-3311 are very similar to those of ProductGh-3353.

The ¹H-¹H COSY data of Product Gh-3311 show that the hydroxymethineproton (δ 5.17 (1H, d, J=9.0 Hz)) is coupled to the oxymethine proton (δ4.71 (1H, d, 0.7=9.0 Hz)) of the epoxy group, with a coupling constant(J) of 9.0 Hz. Since the two vicinal oxymethine protons on an epoxy ringhave a coupling constant (J) normally less than 5 Hz, the couplingconstant of J=8.9 Hz should not represent the coupling of the twovicinal oxymethine protons on the epoxy ring.

The HMBC data of Product Gh-3311 show that: δ 5.17 is correlated with δ160.99 (C-18), δ 159.85 (C-16), δ 106.57 (C-17) and δ 76.98 (C-32); andδ 4.71 is correlated with δ 83.80 (C-33), δ 18.91 (C-34) and δ 67.16(C-31), evidencing that the hydroxy group is connected to C-31, theepoxy group is located at C-32 and C-33, and the side chain attached toC-17 is a 1-hydroxy-2,3-epoxy-3-methylbutyl group.

The NOESY data of Product Gh-3311 show that: δ 7.56 (H-10) is correlatedwith δ 3.56 (H-11); δ 3.56 (H-11) is correlated with δ 2.37 (H₁-21) andδ 1.28 (H₂-21); both δ 2.37 (H₁-21) and δ 1.28 (H₂-21) are correlatedwith δ 2.54 (H-22); δ 2.54 (H-22) is correlated with δ 1.83 (H-25) and δ1.26 (H-24); and δ 5.03 (H-27) is correlated with δ 1.49 (H-29),evidencing that the stereostructure of Product Gh-3311 in this part isidentical to that of gambogic acid, i.e., in a 11S,13R,14S,22Sconfiguration, which includes H-27 and carboxyl group (C-30) in a transrelationship, and a double bond Δ^(27,28) in a Z configuration. Inaddition, since δ 1.49 (H-29) is correlated with δ 1.86 (H₁-20) and δ1.69 (H₂-20), C-2 is in an S configuration.

Based on the above information, product Gh-3311 is identified to be anew compound having the following chemical structure:

Product Gh-3311 is identified by the name “epiformoxanthone G” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11S,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(1-hydroxy-2,3-epoxy-3-methylbutyl)-11-(4-methyl-3-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

32. Product Gh-3272:

Product Gh-3272, which was purified from eluate 3 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 190˜193° C.

EIMS m/z (relative intensity): 626 [M−H₂O]⁺ (2), 598 (2), 545 (100), 517(3), 499 (2), 389 (3), 347 (3), 271 (2), 245 (4), 215 (11), 189 (4), 147(1), 105 (2), 69 (2); HRFABMS [M−H₂O+H]⁺ m/z: 627.2961; calculated forC₃₈H₄₃O₈, 627.2958.

¹H-NMR (600 MHz, CDCl₃): δ 12.71 (1H, s, OH-6), 7.52 (1H, d, J=6.9 Hz,H-10), 6.57 (1H, d, J=10.1 Hz, H-4), 6.03 (1H, dt, J=7.5, 1.3 Hz, H-27),5.59 (1H, d, J=15.7 Hz, H-37), 5.53 (1H, dt, J=15.6, 6.9 Hz, H-36), 5.34(1H, d, J=10.1 Hz, H-3), 5.02 (1H, dt, J=6.2, 1.2 Hz, H-32), 3.46 (1H,dd, J=6.7, 4.6 Hz, H-11), 3.27 (1H, dd, J=14.7, 8.2 Hz, H₁-31), 3.11(1H, br dd, J=14.6, 5.2 Hz, H₂-31), 2.94 (2H, brt, J=5.6 Hz, H-26), 2.49(1H, d, J=9.3 Hz, H-22), 2.36 (1H, dd, J=13.9, 7.2 Hz, H₁-20), 2.29 (1H,dd, J=13.6, 4.8 Hz, H₁-21), 2.25 (1H, dd, J=14.0, 6.5 Hz, H₂-20), 1.72(3H, s, H-34), 1.70 (3H, d, J=1.1 Hz, H-29), 1.67 (3H, s, H-25), 1.63(3H, s, H-35), 1.38 (3H, s, H-19), 1.37 (1H, m, H₂-21), 1.27 (3H, s,H-24), 1.16 (6H, s, H-39, H-40).

¹³C-NMR (150 MHz, CDCl₃): δ 203.30 (C-12), 178.89 (C-8), 170.80 (C-30),161.41 (C-18), 157.41 (C-16), 157.37 (C-6), 142.05 (C-37), 137.79(C-27), 135.37 (C-10), 133.30 (C-9), 131.51 (C-33), 127.80 (C-28),123.91 (C-3), 122.23 (C-32), 120.93 (C-36), 116.30 (C-4), 107.41 (C-17),102.97 (C-5), 100.39 (C-7), 90.90 (C-14), 83.90 (C-23), 83.78 (C-13),80.84 (C-2), 70.61 (C-38), 48.95 (C-22), 46.81 (C-11), 44.67 (C-20),29.83 (C-25), 29.44 (C-39), 29.43 (C-40), 29.27 (C-26), 28.83 (C-24),27.39 (C-19), 25.73 (C-35), 25.14 (C-21), 21.58 (C-31), 20.72 (C-29),18.14 (C-34).

The EIMS data of Product Gh-3272 show [M−H₂O]⁺ (2) at m/z 626 and a basepeak at m/z 545 (100), and the HRFABMS data of Product Gh-3272 show apseudomolecular ion peak [M−H₂O+H]⁺ at m/z 627.2961, indicating thatProduct Gh-3272 has a molecular formula of C₃₈H₄₄O₉, which has sixteenmore mass units than the molecular formula of gambogic acid. As comparedto gambogic acid, the unsaturation number of Product Gh-3272 remainsunchanged.

The ¹H-NMR and ¹³C-NMR data of Product Gh-3272 show that: ProductGh-3272 has a C-2 side chain attached different from that of gambogicacid, namely, a 4-hydroxy-4-methyl-2-pentenyl group in lieu of the4-methyl-3-pentenyl group in gambogic acid.

The ¹H-NMR and ¹³C-NMR data of Product Gh-3272 also reveal that ProductGh-3272 has an oxygen-bearing quaternary carbon (δ 70.61) and twocoupled olefinic protons (δ 5.59 (1H, d, J=15.7 Hz) and δ 5.53 (1H, dt,J=15.6, 6.9 Hz)). In view of the coupling constant (J) of 15.7 Hz, theolefinic group is a trans double bond (i.e., in an E configuration).

The ¹H-¹H COSY data of Product Gh-3272 show that: δ 5.53 (1H, dt,J=15.6, 6.9 Hz) is coupled not only to δ 5.59 (1H, d, J=15.7 Hz), butalso to two methylene protons (δ 2.36 (H₁-20) and δ 2.25 (H₂-20)),evidencing that a trans-disubstituted double bond is located at C-36 andC-37.

The HMBC data of Product Gh-3272 show that: the methylene protons (δ2.25 (H₂-20) and δ 2.36 (H₁-20)) are correlated with δ 27.39 (C-19), δ80.84 (C-2), δ 120.93 (C-36) and δ 142.05 (C-37), as well as an olefiniccarbon (δ 123.91 (C-3)) in a pyran ring; δ 5.53 (H-36) is correlatedwith δ 44.67 (C-20), δ 70.61 (C-38) and δ 142.05 (C-37); and δ 5.59(H-37) is correlated with δ 44.67 (C-20), δ 70.61 (C-38), δ 120.93(C-36), δ 29.44 (C-33) and δ 28.43 (C-40), evidencing that the sidechain attached to C-2 is a 4-hydroxy-4-methyl-2-pentenyl group.

The NOESY data of Product Gh-3272 show that: δ 7.52 (H-10) is correlatedwith δ 3.46 (H-11); δ 3.46 (H-11) is correlated with δ 2.29 (H₁-21) andδ 1.37 (Hz-21); δ 1.37 (H₂-21) is correlated with δ 2.49 (H-22); δ 2.49(H-22) is correlated with a gem-dimethyl group (δ 1.67 (H-25) and δ 1.27(H-24)); and δ 6.03 (H-27) is correlated with δ 1.70 (H-29), evidencingthat the stereostructure of Product Gh-3272 in this part is identical tothat of gambogic acid, i.e., in a 11S,13R,14S,22S configuration, whichincludes H-27 and carboxyl group (C-30) in a trans relationship, and adouble bond Δ^(27,28) in a Z configuration. Besides, the olefinic proton(δ 5.53 (H-36)) is correlated not only with δ 5.59 (H-37) and themethylene protons (δ 2.36 (H₁-20) and δ 2.25 (H₂-20)), but also with thegem-dimethyl group (δ 1.16 (H-39 and H-40)), and another olefinic proton(δ 5.34 (H-3)) is correlated with δ 1.38 (H-19) and δ 2.25 (H₂-20).Since δ 1.38 (H-19) is correlated with δ 1.70 (H-29), C-2 is in an Rconfiguration.

Based on the above information, Product Gh-3272 is identified to be anew compound having the following chemical structure:

Product Gh-3272 is identified by the name “formoxanthone H”{IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11R,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(3-methyl-2-butenyl)-11-(4-hydroxy-4-methyl-2E-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

33. Product Gh-3332:

Product Gh-3332, which was purified from eluate 5 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 102˜106° C.

EIMS m/z (relative intensity): 626 [M−H₂O]⁺ (5), 575 (2), 545 (100), 499(2), 471 (1), 389 (4), 347 (4), 271 (3), 245 (5), 215 (15), 189 (5), 147(2), 105 (3), 69 (2); HRFABMS [M−H₂O+H]⁺ m/z: 627.2966; calculated forC₃₈H₄₃O₈, 627.2958.

¹H-NMR (600 MHz, CDCl₃): δ 12.81 (1H, s, OH-6), 7.55 (1H, d, J=6.9 Hz,H-10), 6.83 (1H, dt, J=7.4, 1.3 Hz, H-27), 6.64 (1H, J=10.0 Hz, H-4),5.68 (1H, d, J=8.4 Hz, H-37), 5.67 (1H, m, H-36), 5.44 (1H, d, J=10.0Hz, H-3), 4.97 (1H, br t, J=6.2 Hz, H-32), 3.48 (1H, dd, J=6.8, 4.5 Hz,H-11), 3.28 (1H, dd, J=14.9, 8.9 Hz, H₁-31), 3.14 (1H, br dd, J=13.5,3.4 Hz, H₂-31), 2.69 (1H, ddd, J=16.4, 6.2, 1.3 Hz, H₁-26), 2.52 (1H, d,J=9.3 Hz, H-22), 2.42 (1H, dd, J=14.3, 4.8 Hz, H₁-21), 2.32 (2H, m,H-20), 2.20 (1H, dd, J=16.3, 8.5 Hz, H₂-26), 1.71 (3H, s, H-34), 1.69(3H, s, H-25), 1.61 (3H, s, H-35), 1.38 (3H, s, H-19), 1.37 (3H, s,H-39), 1.36 (1H, m, H₂-21), 1.33 (3H, s, H-40), 1.30 (3H, s, H-29), 1.27(3H, s, H-24).

¹³C-NMR (150 MHz, CDCl₃): δ 203.38 (C-12), 178.82 (C-8), 169.45 (C-30),161.28 (C-18), 157.69 (C-16), 157.35 (C-6), 141.00 (C-37), 136.22(C-27), 135.59 (C-10), 133.32 (C-9), 131.48 (C-33), 128.43 (C-28),125.06 (C-3), 122.22 (C-32), 121.48 (C-36), 116.22 (C-4), 108.22 (C-17),102.82 (C-5), 100.33 (C-7), 91.06 (C-14), 83.85 (C-23), 83.55 (C-13),80.41 (C-2), 71.65 (C-38), 49.14 (C-22), 46.72 (C-11), 43.61 (C-20),29.84 (C-25), 29.53 (C-39), 29.23 (C-40), 28.81 (C-24), 28.63 (C-26),26.48 (C-19), 25.74 (C-35), 24.95 (C-21), 21.70 (C-31), 18.16 (C-34),11.96 (C-29).

The EIMS data of Product Gh-3332 show [M−H₂O]⁺ (5) at m/z 626 and a basepeak at m/z 545 (100), and the HRFABMS data of Product Gh-3332 show apseudomolecular ion peak [M−H₂O+H]⁺ at m/z 627.2966, indicating thatProduct Gh-3332 has a molecular formula identical to that of productGh-3272, i.e., C₃₈H₄₄O₉, which has 16 more mass units than the molecularformula of gambogic acid or isogambogic acid. As compared to gambogicacid, the unsaturation number of Product Gh-3332 remains unchanged.

The EIMS, ¹H-NMR and ¹³C-NMR data of Product Gh-3332 are generallysimilar to those of Product Gh-3272, implying that Product Gh-3332 mightbe an isomer of Product Gh-3272. Besides, a comparison of the ¹H-NMRdata of a C-20 substituent group (δ 1.33 (3H, s, H-40), δ 1.37 (3H, s,H-39), δ 5.68 (1H, d, J=8.4 Hz, H-37) and δ 5.67 (1H, m, H-36)) ofProduct Gh-3332 with those of Product Gh-3272 reveals that Δ^(36,37) inProduct Gh-3332 is in a Z configuration. Further, as compared to ProductGh-3272, in Product Gh-3332, H-27 has a significant downfield shift(δ_(H) 6.83, Δδ=0.8), and C-29 is influenced by the γ-effect to have anupfield shift (δ_(C) 11.96, Δδ=−8.76). As such, it is presumed thatΔ^(27,28) in product Gh-3332 is in an E configuration.

Based on the above information, Product Gh-3332 is identified to be anew compound having the following chemical structure:

Product Gh-3332 is identified by the name “isoformoxanthone I” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(3-methyl-2-butenyl)-11-(4-hydroxy-4-methyl-2Z-pentenyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2E)-]}.

34. Product Gh-3261:

Product Gh-3261, which was purified from eluate 1 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 103˜105° C.

EIMS m/z (relative intensity): 662 [M]⁺ (15), 647 (5), 603 (2), 545(100), 517 (12), 499 (2), 389 (4), 347 (3), 295 (1), 245 (3), 215 (6),189 (2), 147 (1), 105 (2), 69 (3), 59 (3); HREIMS [M]⁺ m/z 662.3098;calculated for C₃₈H₄₆O₁₀, 662.3091.

¹H-NMR (600 MHz, CDCl₃): δ 12.73 (1H, s, OH-6), 7.49 (1H, d, J=7.0 Hz,H-10), 6.65 (1H, dd, J=10.2, 2.5 Hz, H-4), 5.54 (1H, brt, J=4.6 Hz,H-27), 5.36 (1H, d, J=10.3 Hz, H-3), 5.06 (1H, br s, H-32), 3.50 (1H, t,J=5.7 Hz, H-11), 3.39 (1H, m, H₁-26), 3.36 (1H, d, J=10.1 Hz, H-37),3.27 (1H, dd, J=14.9, 7.4 Hz, H₁-31), 3.22 (1H, br dd, J=14.3, 4.7 Hz,H₂-31), 2.86 (1H, br d, J=14.1 Hz, H₂-26), 2.51 (1H, d, J=9.4 Hz, H-22),2.33 (1H, dd, J=13.5, 4.7 Hz, H₁-21), 2.04 (1H, m, H₁-20), 1.74 (1H, m,H₁-36), 1.72 (3H, s, H-34), 1.68 (1H, m, H₂-20), 1.65 (3H, s, H-25),1.64 (3H, s, H-35), 1.60 (3H, s, H-29), 1.459 (1H, m, H₂-36), 1.456 (3H,s, H-19), 1.39 (1H, dd, J=13.5, 9.6 Hz, H₂-21), 1.27 (3H, s, H-24), 1.21(3H, s, H-39), 1.11 (3H, s, H-40).

¹³C-NMR (150 MHz, CDCl₃): δ 203.34 (C-12), 179.20 (C-8), 168.78 (C-30),161.13 (C-18), 157.75 (C-16), 157.43 (C-6), 136.34 (C-27), 134.74(C-10), 133.91 (C-9), 131.41 (C-33), 128.56 (C-28), 124.18 (C-3), 122.33(C-32), 116.18 (C-4), 107.80 (C-17), 102.49 (C-5), 100.63 (C-7), 90.57(C-14), 84.03 (C-13), 83.64 (C-23), 81.24 (C-2), 78.23 (C-37), 73.70(C-38), 49.01 (C-22), 47.02 (C-11), 38.73 (C-20), 29.92 (C-25), 29.87(C-26), 29.05 (C-24), 27.88 (C-19), 26.70 (C-39), 25.70 (C-35), 25.28(C-36), 25.25 (C-21), 24.28 (C-40), 21.71 (C-31), 20.85 (C-29), 18.22(C-34).

The EIMS data of Product Gh-3261 show a molecular ion peak [M]⁺ at m/z662 and a base peak at m/z 545 (100), and the HREIMS data of ProductGh-3261 show [M]⁺ at m/z 662.3098, indicating that Product Gh-3261 has amolecular formula of C₃₈H₄₆O₁₀, which has thirty-four more mass unitsthan the molecular formula of gambogic acid.

The ¹H-NMR, ¹³C-NMR and HMQC data of Product Gh-3261 show that ProductGh-3261 has a C-2 side chain different from that of gambogic acid,namely, a 3,4-dihydroxy-4-methylpentyl group in lieu of the4-methyl-3-pentenyl group in gambogic acid. Besides, the ¹H-NMR and¹³C-NMR data of Product Gh-3261 reveal that Product Gh-3261 has asecondary hydroxy group (δ 3.36 (1H, d, J=10.1 Hz) and δ 78.23) and anoxygen-bearing quaternary carbon (δ 73.70).

The ¹H-¹H COSY data of Product Gh-3261 show that: a hydroxymethineproton (δ 3.36 (1H, d, J=10.1 Hz)) is coupled to δ 1.459 (1H, m); δ1.459 (1H, m) is coupled to δ 2.04 (1H, m, H₁-20); and four protons oftwo adjacent methylene groups at C-20 and C-36 (δ 2.04 (H₁-20), δ 1.68(H₂-20) and δ 38.73 (C-20); and δ 1.74 (H₁-36), δ 1.459 (H₂-36) and δ25.28 (C-36)] are coupled to each other, indicating that thehydroxymethine proton is H-37. As such, it can be known that a secondaryhydroxy group is attached to C-37 (o 78.23); a tertiary hydroxy group isattached to C-38 (δ 73.70); and C-39 (δ 26.70) and C-40 (δ 24.28) aretertiary methyl groups.

The HMBC data, of Product Gh-3261 show that: the hydroxymethine proton(δ 3.36 (H-37)] is correlated with δ 38.73 (C-20); both δ 2.04 (H₁-20)and δ 1.68 (H₂-20) are correlated with δ 25.28 (C-36), δ 81.24 (C-2) andδ 124.18 (C-3); δ 5.36 (H-3) is correlated with δ 102.49 (C-5), δ 81.24(C-2) and δ 27.88 (C-19); δ 1.456 (H-19) is correlated with δ 81.24(C-2), δ 124.18 (C-3), δ 116.18 (C-4) and δ 38.73 (C-20); δ 1.21 (H-39)is correlated with δ 78.23 (C-37), δ 73.70 (C-38) and δ 24.28 (C-40);and δ 1.11 (H-40) is correlated with δ 78.23 (C-37), δ 73.70 (C-38) andδ 26.70 (C-39), evidencing that the side chain attached to C-2 is a3,4-dihydroxy-4-methylpentyl group.

The NOESY data of Product Gh-3261 show that: δ 7.49 (H-10) is correlatedwith δ 3.50 (H-11); δ 3.50 (H-11) is correlated with δ 2.33 (H₁-21) andδ 1.39 (H₂-21); δ 1.39 (H₂-21) is correlated with δ 2.51 (H-22) and δ1.27 (H-24); δ 2.51 (H-22) is correlated with δ 1.65 (H-25); and δ 5.54(H-27) is correlated with δ 1.60 (H-29), evidencing that thestereostructure of Product Gh-3261 in this part is identical to that ofgambogic acid, i.e., in a 11S,13R,14S,22S configuration, which includesH-27 and carboxyl group (C-30) in a trans relationship, and a doublebond Δ^(27,28) in a Z configuration. In addition, since δ 3.36 (H-37) iscorrelated with δ 1.21 (H-39) and δ 1.11 (H-40), while δ 1.456 (H-19) iscorrelated with δ 1.60 (H-29), C-2 is in an R configuration.

Based on the above information, Product Gh-3261 is identified to be anew compound having the following chemical structure:

Product Gh-3261 is identified by the name “formoxanthone J” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11H,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(3-methyl-2-butenyl)-11-(3,4-dihydroxy-4-methylpentyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

35. Product Gh-3271:

Product Gh-3271, which was purified from eluate 2 of fraction 1, wasdetermined to have the following properties:

Yellow powder; mp 99˜102° C.

EIMS m/z (relative intensity): 662 [M]⁺ (14), 644 (5), 603 (4), 545(100), 517 (15), 499 (3), 419 (4), 389 (6), 347 (5), 283 (5), 245 (6),215 (11), 213 (5), 189 (5), 147 (4), 129 (4), 117 (5), 105 (8), 91 (7),85.9 (12), 83.9 (19), 69 (11), 59 (8), 57 (11), 55 (11); HREIMS [M]⁺m/z: 662.3097; calculated for C₃₈H₄₆O₁₀, 662.3091.

¹H-NMR (600 MHz, CDCl₃): δ 12.75 (1H, s, OH-6), 7.50 (1H, d, J=7.0 Hz,H-10), 6.65 (1H, d, J=10.0 Hz, H-4), 5.46 (1H, d, J=10.0 Hz, H-3), 5.39(1H, ddd, J=10.7, 3.9, 1.4 Hz, H-27), 5.08 (1H, dd, J=7.2, 5.9 Hz,H-32), 3.50 (1H, m, H-11), 3.49 (1H, m, H₁-26), 3.38 (1H, dd, J=10.5,1.9 Hz, H-37), 3.28 (1H, br dd, J=15.1, 5.6 Hz, H₁-31), 3.23 (1H, dd,J=15.0, 7.4 Hz, H₂-31), 2.83 (1H, ddd, J=15.8, 3.9, 2.4 Hz, H₂-26), 2.50(1H, d, J=9.4 Hz, H-22), 2.34 (1H, dd, J=13.5, 4.7 Hz, H₁-21), 2.05 (1H,m, H₁-20), 1.71 (3H, s, H-34), 1.68 (1H, m, H₁-36), 1.64 (3H, s, H-35),1.63 (3H, s, H-25), 1.62 (1H, m, H₂-20), 1.58 (3H, s, H-29), 1.52 (1H,m, H₂-36), 1.44 (3H, s, H-19), 1.37 (1H, dd, J=13.5, 9.5 Hz, H₂-21),1.27 (3H s, H-24), 1.18 (6H, s, H-39, H-40).

¹³C-NMR (150 MHz, CDCl₃): δ 203.26 (C-12), 179.32 (C-8), 168.56 (C-30),160.44 (C-18), 157.89 (C-16), 157.53 (C-6), 135.74 (C-27), 134.69(C-10), 133.85 (C-9), 131.67 (C-33), 129.00 (C-28), 125.28 (C-3), 122.40(C-32), 116.13 (C-4), 108.14 (C-17), 102.95 (C-5), 100.68 (C-7), 90.47(C-14), 84.20 (C-13), 83.57 (C-23), 80.56 (C-2), 78.09 (C-37), 73.63(C-38), 49.07 (C-22), 47.05 (C-11), 36.88 (C-20), 29.99 (C-26), 29.86(C-25), 29.11 (C-24), 26.39 (C-19), 25.89 (C-39), 25.66 (C-35), 25.56(C-36), 25.22 (C-21), 23.40 (C-40), 21.76 (C-31), 20.81 (C-29), 18.07(C-34).

The EIMS data of Product Gh-3271 show a molecular ion peak [M]⁺ at m/z662 and a base peak at m/z 545 (100), and the HREIMS data of productGh-3271 show [M]⁺ at m/z 662.3097, indicating that Product Gh-3271 has amolecular formula of C₃₈H₄₆O₁₀, which has thirty-four more mass unitsthan the molecular formula of gambogic acid.

The ¹H-NMR, ¹³C-NMR and HMQC data of Product Gh-3271 show that ProductGh-3271 has a C-2 side chain different from that of gambogic acid,namely, a 3,4-dihydroxy-4-methylpentyl group in lieu of the4-methyl-3-pentenyl in gambogic acid. Besides, the ¹H-NMR and ¹³C-NMRdata of Product Gh-3271 show that Product Gh-3271 has a secondaryhydroxy group (δ 3.38 (1H, dd, J=10.5, 1.9 Hz) and δ 78.09) and anoxygen-bearing quaternary carbon (δ 73.63).

The ¹H-¹H COSY data of Product Gh-3271 show that: a hydroxymethineproton (δ 3.38 (1H, dd, J=10.5, 1.9 Hz)) is coupled to δ 1.52 (1H, m); δ1.52 (1H, m) is coupled to δ 2.05 (1H, m, H₁-20); and two adjacentmethylene protons (δ 2.05 (H₁-20), δ 1.62 (H₂-20), δ 1.68 (H₁-36), and δ1.52 (H₂-36)) are coupled to each other, evidencing that thehydroxymethine proton is H-37.

The HMQC data of Product Gh-3271 show that: δ 36.88 (C-20), δ 25.56(C-36), and δ 73.63 (C-38), evidencing that the secondary hydroxy groupis attached to C-37, and a tertiary hydroxy group is attached to C-38.

The HMBC data of Product Gh-3271 show that δ 3.38 (H-37) is correlatedwith δ 36.88 (C-20); both δ 2.05 (H₁-20) and δ 1.62 (H₂-20) arecorrelated with δ 25.56 (C-36), δ 78.09 (C-37), δ 80.56 (C-2), δ 125.28(C-3) and δ 26.39 (C-19); δ 5.46 (H-3) is correlated with δ 80.56 (C-2),δ 26.39 (C-19) and δ 102.95 (C-5); δ 1.44 (H-19) is correlated with δ80.56 (C-2), δ 125.28 (C-3), δ 116.13 (C-4) and δ 160.44 (C-18); and δ1.18 (H-39 and H-40) is correlated with δ 78.09 (C-37) and δ 73.63(C-38), evidencing that the side chain attached to C-2 is a3,4-dihydroxy-4-methylpentyl group.

The NOESY data of Product Gh-3271 show that: δ 7.50 (H-10) is correlatedwith δ 3.50 (H-11); δ 3.50 (H-11) is correlated with δ 2.34 (H₁-21) andδ 1.37 (H₂-21); δ 1.37 (H₂-21) is correlated with δ 2.50 (H-22) and δ1.27 (H-24); δ 2.50 (H-22) is correlated with δ 1.63 (H-25); and δ 5.39(H-27) is correlated with δ 1.58 (H-29), evidencing that thestereostructure of Product Gh-3271 in this part is identical to that ofgambogic acid, i.e., in a 11S,13R,14S,22S configuration, which includesH-27 and carboxyl group (C-30) in a trans relationship, and a doublebond Δ^(27,28) in a Z configuration. In addition, since δ 3.38 (H-37) iscorrelated with δ 1.18 (H-39 and H-40), and δ 2.05 (H₁-20) is correlatedwith δ 1.58 (H-29), C-2 is in an S configuration.

Based on the above information, Product Gh-3271 is identified to be anew compound having the following chemical structure:

Product Gh-3271 is identified by the name “epiformoxanthone J” {IUPACnomenclature: [2-butenoic acid,2-methyl-4-[(1R,3aS,5S,11S,14aS)-3a,4,5,7-tetrahydro-8-hydroxy-3,3,11-trimethyl-13-(3-methyl-2-butenyl)-11-(3,4-dihydroxy-4-methylpentyl)-7,15-dioxo-1,5-methano-1H,3H,11H-furo[3.4-g]pyrano[3.2-b]xanthen-1-yl]-(2Z)-]}.

Conclusion:

According to the experimental results obtained above, the thirty-fiveproducts purified from fractions 1-3 are found to include 17 newcompounds and 18 known compounds, as summarized in the following Table4. Inasmuch as Products Gh-47, Gh-631, Gh-4601, Gh-4602 and Gh-2301 arefound to be known compounds, the in vitro anti-cancer activities ofwhich had been tested in U.S. Pat. No. 7,138,428 B2, these five productsare not tested in the following pharmacological experiments.

TABLE 4 Thirty-five products purified from fractions 1-3 Product no.Nomenclature New compounds Gh-2607-B Formoxanthone B Gh-2607-1AEpiformoxanthone B Gh-2508 β-Gambogellic acid Gh-2507 β-Epigambogellicacid Gh-1631 Formoxanthone C Gh-1050 3α-Hydroxygambogellic acid Gh-3291Formoxanthone D Gh-3352 Formoxanthone E Gh-3351 Epiformoxanthone EGh-1052 Formoxanthone F Gh-1036 Epiformoxanthone F Gh-3353 FormoxanthoneG Gh-3311 Epiformoxanthone G Gh-3272 Formoxanthone H Gh-3332Isoformoxanthone I Gh-3261 Formoxanthone J Gh-3271 Epiformoxanthone JKnown compounds Gh-47 Isomorellic acid Gh-631 Formoxanthone A Gh-4601Isomorellinol Gh-4602 Morellic acid Gh-2301 Desoxymorellin Gh-4301Desoxygambogenin Gh-2605 Gambogic acid Gh-2606 Eepigambogic acid Gh-1641Isogambogic acid Gh-1642 Epiisogambogic acid Gh-2603-2 Gambogellic acidGh-2603-1 Epigambogellic acid Gh-2501 Isomorellin Gh-2505 Gambogenicacid Gh-2642 Isogambogenic acid Gh-1601-A 30-Hydroxygambogic acidGh-1602 30-Hydroxyepigambogic acid Gh-2641-1 Neogambogic acid

Example 4 Pharmacological Experiment of the Purified Compounds Obtainedfrom the Product TSB-14

In order to explore the possible biological activities of the purifiedproducts obtained in the above Example 2 (excluding Products Gh-47,Gh-631, Gh-4601, Gh-4602 and Gh-2301), the following pharmacologicalexperiment was performed by the MDS Pharma Services.

In Vitro Anti-Cancer Test

The in vitro anti-cancer test was primarily used to detect the effect ofa candidate drug on cancer cell proliferation. The working principleinvolved therein is the ability of viable cells to shift alamarBlue (AbDSerotec, UK) from its originally non-fluorescent oxidized state(non-fluorescent, blue) to a reduced form (fluorescent, red) havingfluorescence via metabolic reaction. According to the fluorescent dataresult generated by the alamarBlue reagent thus obtained, theproliferation of the viable cells and cell activity can be quantifiedfor detection.

In the in vitro anti-cancer tests, a candidate drug was tested at 5different concentrations of 0.01, 0.1, 1, 10, and 100 μg/ml, and sixhuman cancer cell lines and a normal human cell line were used (seeTable 5). In addition, 40% dimethylsulfoxide (DMSO) was used as a normalcontrol, and mitomycin was used as a positive control.

TABLE 5 Cell lines used in the in vitro anti-cancer tests Cell lineSource Human breast adenocarcinoma cell MCF7 ATCC HTB-22 Human colonadenocarcinoma cell HT-29 ATCC HTB-38 Human promyelocytic leukemia cellHL-60 ATCC CCL-240 Human hepatocellular carcinoma cell HepG2 ATCCHB-8065 Human lung carcinoma cell A549 ATCC CCL-185 Human histocyticlymphoma cell U937 ATCC CRL-1593 Human umbilical venal epithelial cell(HUVEC) ATCC CRL-1730

According to the experimental reports provided by the MDS PharmaServices, IC₅₀ (50% inhibition concentration) and LC₅₀ (50% lethalconcentration) of the thirty purified products obtained in the aboveExample 2 are shown in Tables 6-7, respectively.

TABLE 6 IC₅₀ (μg/mL) of thirty purified products obtained in Example 2with respect to cancer cells Tested Cell line product MCF-7 HT-29 HL-60HepG2 A549 U937 HUVEC Gh-3353 2.4 5.0 1.3 5.3 2.6 4.8 5.5 Gh-3271 0.161.8 3.0 0.46 0.25 0.11 0.42 Gh-3311 8.0 7.9 1.4 7.9 5.0 1.9 4.0 Gh-32610.36 1.1 1.9 1.0 0.19 0.11 0.29 Gh-3351 0.99 1.9 11.7 2.9 0.77 0.24 0.11Gh-3272 0.73 0.36 1.7 1.2 1.7 0.16 0.12 Gh-3332 0.87 0.85 11.8 6.7 1.30.23 0.11 Gh-3291 0.45 0.081 4.5 0.65 0.62 0.22 0.34 Gh-2507 0.19 1.60.20 1.7 0.13 0.45 0.41 Gh-2508 0.087 1.8 0.28 1.9 0.16 0.28 0.52Gh-1050 0.14 4.8 0.17 1.1 0.13 0.21 0.78 Gh-1631 0.14 6.1 0.19 0.85 0.140.18 0.58 Gh-2641-1 0.20 2.6 0.18 1.7 0.15 0.26 0.54 Gh-1036 0.35 1.40.45 0.88 0.22 0.29 0.46 Gh-1052 0.52 1.6 0.23 1.2 0.46 0.22 0.47Mitomycin (μM) 0.79 0.099 0.13 0.47 0.35 0.18 0.37 Gh-2505 1.9 2.1 0.752.1 0.62 1.1 0.28 Gh-2501 1.3 2.1 1.0 2.2 0.61 0.90 0.26 Mitomycin (μM)0.069 0.39 0.048 0.053 0.11 0.050 0.14 Gh-4301 19 13 3.5 12 0.51 11 0.29Mitomycin (μM) 0.089 0.50 0.078 0.069 0.097 0.059 0.075 Gh-2603-1 2.21.6 1.0 1.0 0.036 1.3 0.26 Gh-2603-2 2.2 1.4 0.93 0.65 0.20 1.5 0.14Gh-2605 4.4 0.18 0.17 0.23 0.12 0.76 0.098 Gh-2606 1.3 0.39 0.66 0.380.10 0.41 0.11 Gh-2607-B + 1.4 0.94 0.90 1.1 0.091 0.84 0.15 Gh-2607-1AMitomycin (μM) 0.15 0.49 0.098 0.11 0.099 0.072 0.12 Gh-1641 1.7 1.4 8.00.86 0.47 9.4 0.18 Gh-1642 2.4 1.9 6.0 1.1 0.51 8.8 0.18 Mitomycin (μM)0.080 0.51 0.062 0.075 0.15 0.098 0.10 Gh-2642 3.0 12 9.0 11 1.5 1.3 2.4Mitomycin (μM) 0.13 0.57 0.12 0.062 0.24 0.17 0.13 Gh-1602 0.27 0.94 1.01.3 0.40 0.14 0.25 Mitomycin (μM) 0.079 0.37 0.20 0.073 0.21 0.19 0.088Gh-2607-B 0.10 0.30 0.13 0.31 0.10 0.10 0.07 Gh-2607-1A 0.12 0.24 0.490.43 0.19 0.15 0.43 Gh-1601-A 0.72 0.17 0.56 1.3 0.11 0.09 0.19 Gh-33520.075 0.18 0.26 0.16 0.18 0.11 0.084 Mitomycin (μM) 0.15 0.11 0.24 0.210.098 0.18 0.15

TABLE 7 LC₅₀ (μg/mL) of thirty purified products obtained in Example 2with respect to cancer cells Tested Cell line product MCF-7 HT-29 HL-60HepG2 A549 U937 HUVEC Gh-3353 9.6 20.8 2.4 35.5 8.2 14.6 8.4 Gh-32710.62 5.8 17.1 4.6 0.51 0.35 1.5 Gh-3311 14.0 38.0 2.2 25.6 21.2 5.4 13.9Gh-3261 2.8 3.9 7.0 2.8 0.50 0.31 1.4 Gh-3351 3.8 72.6 22.1 31.2 3.40.62 3.3 Gh-3272 2.8 6.6 5.1 3.7 4.7 0.43 2.5 Gh-3332 3.3 73.8 30.5 19.64.6 1.4 3.3 Gh-3291 2.8 4.7 30.0 8.7 3.3 0.61 1.4 Gh-2507 2.2 8.6 5.37.1 0.53 2.7 21.0 Gh-2508 1.7 5.5 6.6 5.9 0.46 2.2 5.4 Gh-1050 1.0 7.77.0 3.8 0.74 2.3 3.4 Gh-1631 0.87 8.3 4.1 3.7 0.20 0.86 2.3 Gh-2641-13.5 6.8 4.5 4.6 0.22 1.0 4.3 Gh-1036 3.8 12.0 3.1 8.6 0.52 0.81 4.8Gh-1052 2.4 5.7 3.4 4.0 2.5 0.69 3.1 Mitomycin (μM) >10 8.9 1.3 4.7 4.00.75 8.3 Gh-2505 19 4.4 7.2 6.5 12 3.7 2.3 Gh-2501 3.7 4.0 4.0 4.5 2.13.1 1.8 Mitomycin (μM) 6.4 5.6 0.81 1.8 2.0 1.1 1.4 Gh-4301 56 42 >10051 6.2 >100 1.3 Mitomycin (μM) 5.8 7.8 2.5 2.6 2.1 3.3 8.5 Gh-2603-1 4.23.7 2.9 3.3 0.34 3.1 1.7 Gh-2603-2 4.2 3.9 2.4 3.5 0.47 3.6 1.7 Gh-26055.9 0.61 2.9 3.5 0.37 2.9 1.6 Gh-2606 1.6 2.0 2.4 3.0 0.31 3.1 1.4Gh-2607-B + 2.2 2.1 2.7 2.5 0.51 2.7 1.4 Gh-2607-1A Mitomycin (μM) 1.89.3 1.3 1.3 1.9 2.0 6.6 Gh-1641 33 4.4 32 4.5 3.0 36 1.3 Gh-1642 29 4.835 5.3 2.8 33 1.3 Mitomycin (μM) 5.8 8.5 1.2 2.3 3.6 1.9 2.4 Gh-2642 1042 32 29 4.6 4.1 11 Mitomycin (μM) >10 >10 2.3 1.6 4.5 0.87 2.4 Gh-16024.0 4.2 3.3 3.5 3.7 0.69 0.94 Mitomycin (μM) >10 >10 1.8 2.4 5.3 0.821.7 Gh-2607-B 0.68 1.0 3.1 4.0 0.33 0.25 1.6 Gh-2607-1A 1.5 0.84 2.6 3.90.45 0.37 1.5 Gh-1601-A 4.4 0.77 3.0 4.1 6.6 0.28 5.4 GH-3352 1.8 0.680.93 2.0 0.99 0.26 0.85 Mitomycin (μM) 17 22 0.89 5.2 4.4 0.85 1.8

It can be known from the results shown in Tables 6-7 that the seventeennew compounds obtained in the above Example 2 all have significanteffects in inhibiting the growth of tumor/cancer cells and killingtumor/cancer cells. Therefore, the seventeen new compounds obtained inthe above Example 2 all have high potentials for serving as anti-cancerdrugs.

All patents and literature references cited in the present specificationare hereby incorporated by reference in their entirety. In case ofconflict, the present description, including definitions, will prevail.

While the invention has been described with reference to the abovespecific embodiments, it is apparent that numerous modifications andvariations can be made without departing from the scope and spirit ofthis invention. It is therefore intended that this invention be limitedonly as indicated by the appended claims.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. A method of inhibiting thegrowth of tumor/cancer cells wherein the tumor/cancer cells comprise oneor more of: human breast adenocarcinoma cells, human colonadenocarcinoma cells, human promyelocyte leukemia cells, humanhepatocellular carcinoma cells, human lung carcinoma cells, or humanhistocytic lymphoma cells, the method comprising contacting the cellswith one or more compounds selected from the group consisting of: (1) acompound of the formula:

and, (2) a compound of the formula:


5. (canceled)